Dipeptidyl peptidase IV inhibitors and their uses as anti-cancer agents

ABSTRACT

The present invention provides new uses of DPIV-inhibitors of the present invention, and their corresponding pharmaceutically acceptable acid addition salt forms, for treating conditions mediated by DPIV or DPIV-like enzymes, such as cancer and tumors. In a more preferred embodiment, the compounds of the present invention are useful for the treatment of metastasis and tumor colonization.

RELATED APPLICATIONS

This application is a continuation of Ser. No. 10/172,809 filed Jun. 13,2002, now abandoned.

This application claims the priority of U.S. provisional applicationU.S. 60/301,158 entitled Peptide Structures Useful for Competitivemodulation of Dipeptidyl Peptidase IV Catalysis filed on Jun. 27, 2001.Priority is also claimed from U.S. provisional application U.S.60/360,909 entitled Glutaminyl-based DPIV Inhibitors filed on Feb. 28,2002. This application also claims the priority of the following foreignapplications EP 01 114 796.4 entitled Peptide Structures Useful forCompetitive Modulation of Dipeptidyl Peptidase IV Catalysis having apriority date of Jun. 27, 2001, DE 101 50 203.6 entitled Peptidylketoneals Inhibitoren der DPIV having a priority date of Oct. 12, 2001 and DE101 54 689.0 entitled Substituierte Aminoketonverbindungen having apriority Date of Nov. 9, 2001. The above applications are incorporatedin their entirety by reference.

FIELD OF THE INVENTION

The present invention relates to inhibitors of dipeptidyl peptidase IVand dipeptidyl peptidase IV-like enzyme activity and, more particularly,pharmaceutical compositions containing said compounds, and the use ofsaid compounds for the treatment of cancer and tumors. The presentinvention especially provides a method for the inhibition of metastasisand tumor colonization.

BACKGROUND ART

Dipeptidyl peptidase IV (DPIV) is a serine protease which cleavesN-terminal dipeptides from a peptide chain containing, preferably, aproline residue in the penultimate position. Although the biologicalrole of DPIV in mammalian systems has not been completely established,it is believed to play an important role in neuropeptide metabolism,T-cell activation, attachment of cancer cells to the endothelium and theentry of HIV into lymphoid cells.

Likewise, it has been discovered that DPIV is responsible forinactivating glucagon-like peptide-1 (GLP-1) and glucose-dependentinsulinotropic peptide also known as gastric-inhibitory peptide (GIP).Since GLP-1 is a major stimulator of pancreatic insulin secretion andhas direct beneficial effects on glucose disposal, in WO 97/40832 andU.S. Pat. No. 6,303,661 inhibition of DPIV and DPIV-like enzyme activitywas shown to represent an attractive approach for treatingnon-insulin-dependent diabetes mellitus (NIDDM).

The present invention provides a new use of DPIV-inhibitors for thetreatment of conditions mediated by inhibition of DPIV and DPIV-likeenzymes, in particular the treatment of cancer and tumors and theinhibition of metastasis and tumor colonization, and pharmaceuticalcompositions e.g. useful in inhibiting DPIV and DPIV-like enzymes and amethod of inhibiting said enzyme activity.

This invention relates to a method of treatment, in particular to amethod for the treatment of cancer, tumors, metastasis and tumorcolonization and to compositions for use in such method. Dipeptidylpeptidase IV (DPIV) is a post-proline (to a lesser extent post-alanine,post-serine or post-glycine) cleaving serine protease found in varioustissues of the body including kidney, liver, and intestine.

It is known that DPIV-Inhibitors may be useful for the treatment ofimpaired glucose tolerance and diabetes mellitus (International PatentApplication, Publication Number WO 99/61431, Pederson R A et al,Diabetes. 1998 August; 47(8):1253–8 and Pauly R P et al, Metabolism 1999March; 48(3):385–9). In particular WO 99/61431 discloses DPIV-Inhibitorscomprising an amino acid residue and a thiazolidine or pyrrolidinegroup, and salts thereof, especially L-threo-isoleucyl thiazolidine,L-allo-isoleucyl thiazolidine, L-threo-isoleucyl pyrrolidine,L-allo-isoleucyl thiazolidine, L-allo-isoleucyl pyrrolidine, and saltsthereof.

Further examples of low molecular weight dipeptidyl peptidase IVinhibitors are agents such as tetrahydroisoquinolin-3-carboxamidederivatives, N-substituted 2-cyanopyroles and -pyrrolidines,N-(N′-substituted glycyl)-2-cyanopyrrolidines, N-(substitutedglycyl)-thiazolidines, N-(substituted glycyl)-4-cyanothiazolidines,amino-acyl-borono-prolyl-inhibitors, cyclopropyl-fused pyrrolidines andheterocyclic compounds. Inhibitors of dipeptidyl peptidase IV aredescribed in U.S. Pat. No. 6,380,398, U.S. Pat. No. 6,011,155; U.S. Pat.No. 6,107,317; U.S. Pat. No. 6,110,949; U.S. Pat. No. 6,124,305; U.S.Pat. No. 6,172,081; WO 95/15309, WO 99/61431, WO 99/67278, WO 99/67279,DE 198 34 591, WO 97/40832, DE 196 16 486 C 2, WO 98/19998, WO 00/07617,WO 99/38501, WO 99/46272, WO 99/38501, WO 01/68603, WO 01/40180, WO01/81337, WO 01/81304, WO 01/55105, WO 02/02560 and WO 02/14271, theteachings of which are herein incorporated by reference in theirentirety.

The term DPIV-like enzymes relates to structurally and/or functionallyDPIV/CD26-related enzyme proteins (Sedo & Malik, Dipeptidyl peptidaseIV-like molecules: homologous proteins or homologous activities?Biochimica et Biophysica Acta 2001, 36506: 1–10). In essence, this smallgroup of enzymes has evolved during evolution to releaseH-Xaa-Pro-Dipeptides and H-Xaa-Ala-Dipeptides from N-terminus of oligo-or polypeptides. They show the common feature, that they accomotate inthe Pro-position also Al, Ser, Thr and other amino acids with smallhydrophobic side-chains as, Gly or Val. The hydrolytic efficacy isranked Pro>Ala>>Ser, Thr>>Gly, Val. Same proteins have been onlyavailable in such small quantities, that only the post-Pro or post-Alacleavage could be established. While the proteins: DPIV, DP II, FAPα(Seprase), DP 6, DP 8 and DP 9 are structurally related and show a highsequence homology, attractin is an extraordinary functional DPIV-likeenzyme, characterized by a similar activity and inhibitory pattern.

Further DPIV-like enzymes are disclosed in WO 01/19866, WO 02/04610, WO02/34900 and WO 02/31134. WO 01/19866 discloses novel human dipeptidylaminopeptidase (DPP8) with structural und functional similarities toDPIV and fibroblast activation protein (FAP). WO 02/04610 providesreagents, which regulate human dipeptidyl peptidase IV-like enzyme andreagents which bind to human dipeptidyl peptidase IV-like enzyme geneproduct. These reagents can play a role in preventing, ameliorating, orcorrecting dysfunctions or diseases including, but not limited to,tumors and peripheral and central nervous system disorders includingpain and neurodegenerative disorders. The dipeptidyl peptidase IV-likeenzyme of WO 02/04610 is well known in the art. In the Gene Bank database, this enzyme is registered as KIAA1492 (registration in February2001, submitted on Apr. 4, 2000, AB040925). In the Merops data base, thedipeptidyl peptidase IV-like enzyme of WO 02/04610 is registered asnon-protease homologue. The Merops homologue of the dipeptidyl peptidaseIV-like enzyme disclosed in WO 02/04610 and the active site motivethereof was confirmed by the human genome project. WO 02/34900 disclosesa novel dipeptidyl peptidase 9 (DPP9) with significant homology with theamino acid sequences of DPIV and DPP8. WO 02/31134 discloses threeDPIV0-like enzymes. DPRP1, DPRP2 and DPRP3. Sequence analysis revealed,that DPRP1 is identical to DPP8, as disclosed in WO 01/19868, that DPRP2is identical to DPP9 and that DPRP3 is identical to KIAA1492 asdisclosed in WO 02/04610.

DPIV and DPIV-Like Enzymes in Immunophysiology and Cancer

Dipeptidyl peptidase IV (DPIV; EC 3.4.14.5; CD26) CD26 is a M r 110,000surface glycoprotein with an array of diverse functional properties thatis expressed on a number of tissues, including epithelial cells andleukocyte subsets (Mentlein, 1999). Furthermore, it is amembrane-associated ectopeptidase that possesses DPIV-like activity inits extracellular domain and is able to cleave N-terminal dipeptidesfrom polypeptides with either L-proline or L-alanine in the penultimateposition. In general, DPIV is recognized as an ectopeptidase with atriple functional role. DPIV is involved in catalyzing the release ofXaa-Pro dipeptides from circulating hormones and chemokines (De Meesteret al, 1999; Mentlein, 1999), in T cell dependent immune responses(Kähne et al, 1999; Korom et al, 1997), and in cell adhesion includingmetastasis (Mentlein, 1999).

In addition DPIV has been identified as the ADA binding protein, therebyregulating ADA surface expression, with the DPIV/ADA complex perhapsplaying a key role in the catalytic removal of local adenosine toregulate immune system function. Besides being a key immunoregulatorymolecule, DPIV may have a potential role in the development of certainneoplasms (Mattern et al., 1993; Carbone et al., 1995). In eukaryoticcells, cell cycle progression is controlled at the G1-S checkpoint by agroup of related enzymes known as the CDKs, which are positivelyregulated by their physical association with regulatory subunits calledcyclins. It has been demonstrated that binding of soluble anti-CD26antibodies inhibits the growth of anaplastic large cell T-cell lymphomacell lines, both in in vitro and in vivo experiments (Ho et al., 2001).

Cancer Pathomechanisms

Cancer is a group of over 150 diseases characterized by the uncontrolledgrowth of abnormal cells in the body. Normal cells can become abnormalwhen they are exposed to carcinogens such as radiation or particulardrugs or chemicals. They can also turn malignant (cancerous) when theyare attacked by certain viruses or when some not-yet-fully-understoodinternal signal occurs. Once cells become malignant, they multiply morerapidly than usual. Then they often form masses called tumors thatinvade nearby tissue and interfere with normal bodily functions. Cancercells also have a tendency to spread to other parts of the body, wherethey may form a secondary tumor.

Mechanisms of Metastasis

The outcome of cancer metastasis depends on multiple interactions withinthe target tissue and depends on the microenvironment including cellularadhesion molecules (Carlos, 2001), chemokines (Muller et al., 2001), orhydrodynamic effects (Haier and Nicholson, 2001) and many other factors(Fidler, 2001). In addition, a very rapid attraction of leukocytes andspecific cellular responses at the tumor sites may play a critical rolein the early host defense against cancer (Shingu et al.; 2002). Theseearly changes may be of critical importance for the outcome ofmetastatic disease and may extend the present understanding of the hostresistance against metastasis.

DPIV and DPIV-Like Enzymes and Tumor Adhesion and Colonization

For cancer cell or metastatic cell adhesion, a prominent expression ofDPIV on endothelia of lung capillaries accounts for arrest of bloodborne breast cancer cells (Johnson et al, 1993). Fibronectin (FN) andprobably also collagen collected on the breast cancer cell surface wereidentified as the principal ligands for DPIV (Abdel-Ghany et al, 1998;Cheng et al, 1998).

Ho and colleagues (2001) show that binding of soluble anti-CD26monoclonal Ab 1F7 inhibits the growth of the human CD301 anaplasticlarge cell T-cell lymphoma cell line Karpas 299 in both in in vitro andin vivo experiments. In vitro experiments show that 1F7 induces cellcycle arrest at the G1-S checkpoint, associated with enhanced p21expression that is dependent on de novo protein synthesis. Furthermore,experiments with a severe combined immunodeficient mouse tumor modeldemonstrate that 1F7 treatment significantly enhances survival oftumor-bearing mice by inhibiting tumor formation.

Protease Inhibitors, Antibodies and Proteases as Anti-Tumor Agents

WO 95/29691 discloses proline phosphonate derivatives as inhibitors ofserine proteases with chymotrypsin-like, trypsin-like, elastase-like anddipeptidyl peptidase IV specificity and their roles as anti-inflammatoryagents, anticoagulants, anti-tumor agents and anti-AIDS agents.

WO 98/53812 and WO 97/48409 disclose novel methods of using phosphonatederivatives, hydroxyphosphinyl derivatives, and phosphoramidatederivatives to inhibit N-Acetlyated α-Linked Acidic Dipeptidase(NAALADase) enzyme activity, and to treat prostate diseases, especiallyusing these compounds for the inhibition of prostate cancer cell growth.

WO 01/92273 discloses new benzenedicarboxylic acid derivative compounds,pharmaceutical compositions, diagnostic methods and diagnostic kits thatinclude those compounds and methods of using those compounds forinhibiting NAALADase enzyme activity, detecting diseases where NAALADaselevels are altered, affecting neuronal activity, affecting TGF-βactivity, inhibiting angiogenesis and treating glutamate abnormalities,neuropathy, pain, compulsive disorders, prostate diseases, cancer andglaucoma.

WO 01/34596 discloses pyrrolecarbonylimino derivatives, pharmaceuticalcompounds and methods of using those compounds to inhibit NAALADaseenzyme activity, thereby affecting neuronal activities, inhibitingangiogenesis and treating glutamate abnormalities, compulsive disorders,prostate diseases and cancer.

WO 00/71135 discloses a method for treating subjects with abnormal cellproliferation. The method involves administering to subjects in need ofsuch treatment an effective amount of boro-proline compounds, to inhibitcell proliferation such as that associated with tumor growth andmetastasis. A method for inhibiting angiogenesis in an abnormalproliferative cell mass by the administration of a boro-prolinederivative is also provided. The invention of WO 00/71135 is based, inpart, on the observation, that the boro-proline derivatives are able toinhibit the enzymatic activity of fibroblast activation protein-alpha(FAP-α).

WO 00/71571 relates to a prodrug that is capable of being converted intoa drug by the catalytic action of human fibroblast activationprotein-alpha (FAP-α), said prodrug having a cleavage site which isrecognised by FAP-α, and said drug being cytotoxic or cytostatic underphysiological conditions. These prodrugs are converted into a drug atthe site of the tumor.

WO 00/10549 discloses compounds and a method for regulation of substrateactivity in vivo useful for the treatment of medical disorders such asarteriosclerosis, allergies, inflammation, angiogenesis, cardiogenesis,neoplasm, tumor, cancer, a hepatic disease, an intestinal disease, organvascularization, and microbial and viral infections. The compoundsconsist of a targeting moiety that binds to DPIV, and a reactive group,that reacts at a reactive center of DPIV. Said compounds are used toprevent chemokine alteration by inhibiting DPIV activity.

WO 00/36420 discloses a method for identifying nucleotide sequences thatare differentially expressed in tumor cells, preferably primary breasttumor cells, comprising exposing a tumor cell containing tissue sampleto an agent specific for fibroblast activation protein (FAP) separatingcells recognised by said agent from the remaining cells in the sampleand harvesting said remaining cells. Nucleic acid molecules derived fromthe use of this technique are also described, together with compositionscomprising the same and their uses in pharmaceutical compositions fortreating a disease, preferably breast cancer.

WO 99/47152 discloses a method of suppressing the malignant phenotype orinducing apoptosis of cancer cells in a subject, comprising introducinginto the cancer cell an amount of a nucleic acid encoding a dipeptidylpeptidase IV protein or fibroblast activating protein-α, therebysuppressing the malignant phenotype of the cancer. WO 99/47152 alsodiscloses a method of inducing expression of dipeptidyl peptidase IV orfibroblast activating protein-α in cancer cells of a subject, comprisingadministering to the subject a pharmaceutical composition comprising atherapeutically effective amount of an agent capable of activatingtranscription of the dipeptidyl peptidase IV gene or fibroblastactivating protein-α gene and a pharmaceutical acceptable carrier ordiluent.

WO 01/74299 discloses antibodies that specifically bind to a membraneprotease complex, the complex consisting of two homodimers of sepraseand dipeptidyl peptidase IV (DPIV), obtained from mammalian, preferablyhuman cell membranes. The antibodies specifically bind to the DPIVprotease of the seprase-DPIV complex. This membrane protease complexresides on cell surface invadopodia at the leading edge of angiogenicendothelia, migratory fibroblasts, and invading cancer cells.

WO 02/20825 relates to novel methods and compositions for detection andisolation of cancer cells with metastatic potential. WO 02/20825 furtherrelates to assays for measuring the metastatic potential of such cancercells and drug screening assays for the indentification of agents havinganti-metastatic potential. Also disclosed are methods and compositionsfor inhibiting the metastatic potential of cancer cells by modulatingthe activity of serine integral membrane proteases [(SIMP) consisting ofseprase and dipeptidyl peptidase IV (DPIV)] expressed on the surface ofmetastasizing cancer cells, by using antibodies against SIMP.

Current Treatments of Cancer and Tumor Cell Adhesion

Current cancer treatment regimens comprise surgery, chemotherapy,radiation therapy, and other treatment methods including immunotherapy.Immunotherapy is composed of the usage or the modification of intrinsicbodily mechanisms—in most cases immune mechanisms—to fight cancer.Chemotherapy kills cancer cells through the use of drugs or hormones.Taken either orally or through injection, chemotherapeutic agents areused to treat a wide variety of cancer. They may be given alone or incombination with surgery or radiation or both. Chemotherapy is anestablished way to destroy hard-to-detect cancer cells that have spreadand are circulating in the body. Anemia (low number of red blood cells)is a frequent side effect of chemotherapy and may cause symptoms such asextreme tiredness, dizziness, or shortness of breath. Epoetin alfa(Procrit®, Epogen®)—recombinant erythropoietin that stimulates red bloodcell production—is a prescription drug available for the treatment ofchemotherapy-related anemia.

Immunotherapy uses the body's own immune system or other parts of theorganism to destroy cancer cells. This form of treatment is still beingintensively studied in clinical trials; it is not yet widely availableto most cancer patients. The various immunological agents used includesubstances produced by the body (such as the interferons, theinterleukins and tumor necrosis factor) and laboratory-producedsubstances (such as monoclonal antibodies and vaccines). Immunologicalagents work in different ways and can be used independently or incombination with other forms of treatment.

Angiogenesis Inhibitors as Anti-Metastatic Drugs in Immunotherapy

Angiogenesis inhibitors are drugs that block the development of newblood vessels. Solid tumors cannot grow without inducing the formationof new blood vessels. Blocking the development of new blood vessels cutsoff the tumor's supply of oxygen and nutrients.

Several angiogenesis inhibitors are currently being tested in humantrials. In cancerous tissue, tumors cannot grow or spread (metastasize)without the development of new blood vessels. Blood vessels supplytissues with oxygen and nutrients necessary for survival and growth.

SUMMARY OF THE INVENTION

The present invention provides new uses of DPIV-inhibitors of formulas 1to 12, and their corresponding pharmaceutically acceptable acid additionsalt forms for treating cancer and tumors. In a more preferredembodiment, the compounds of the present invention are useful for theprevention and inhibition of metastasis and tumor colonization.

Reduced expression of the ectopeptidase DPIV and lack of DPIV-likeactivity in lungs of mutant F344 rats lacking DPIV enzymic activity andexpression results in reduced adhesion of cancer cells and in reducedlung metastasis. In vivo cell adhesion and growth of the F344 ratsyngeneic mammary adenocarcinoma MADB106 was studied in F344 rats afteracutel and chronic treatment with DPIV-ligands in vivo. Mutant F344substrains lacking DPIV enzymic activity and wild-type-like F344 weretested. Chronic intragastric infusion of isoleucyl cyano pyrrolidine TFAand isoleucyl thiazolidine fumarate via osmotic minipumps over two weeksdose-dependently reduced the cancer-induced weight loss and the numberof tumor colonies on the lung surface. Thus, metastasis of MADB106 isreduced by chronic treatment using different DPIV Inhibitors (isoleucylthiazolidine fumarate; isoleucyl cyano pyrrolidine TFA) suggestingprotective-like class effects by the two differentDPIV-inhibitor/ligands. Possibly, isoleucyl thiazolidine fumarate andisoleucyl cyano pyrrolidine TFA protect from metastasis either viainteraction with cell adhesion processes, via a modification of thecellular host defense mechanisms, via modulation of angiogenesis, viadirect effects on cancer cells, or via increased levels of DPIVsubstrates, which indirectly mediate protective-like effects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Effect of single injection of isoleucyl thiazolidine fumarate onlung metastasis in F344 rats. Vital dye (Carboxyfluorescein; CFSE)labeled MADB106 tumor cells were injected via the lateral tail vein andlungs were collected 30 min after inoculation. CFSE positive tumor cellsin lungs were quantified by means of immunohistology and image analysis.Data represent means±SEM; no significant differences vs. saline treatedcontrols were found.

FIG. 2: Effect of single injection of isoleucyl thiazolidine fumarate ontumor cell adhesion 30 min after injection in F344 substrains mutant forDPIV. CFSE labeled MADB106 tumor cells were injected via the lateraltail vein and lungs were collected 30 min after inoculation. Note thepromoting effect in mutant F344GER and F344JAP rats in contrast to thelack of effects in wild-type F344 rats. Data represent means±SEM;*p<0.05 reflecting significant differences vs. wild-type F344USA animalsdetermined by ANOVA and Fisher PLSD.

FIG. 3: Effect of single injection of isoleucyl cyanopyrrolidine TFA ontumor cell adhesion 30 min after injection in F344USA rats. CFSE labeledMADB106 cancer cells were injected via the lateral tail vein and lungswere collected 30 min after inoculation. CFSE positive tumor cells inlungs were quantified, by means of immunohistology and image analysis.Data represent means±SEM; significant differences vs. saline treatedcontrols were not found.

FIG. 4: Effect of single injection of valyl pyrrolidine fumarate ontumor cell adhesion 30 min after injection in F344USA rats. CFSE labeledof MADB106 adenocarcinoma cells were injected via the lateral tail veinand lungs were collected 30 min after inoculation. CFSE positive tumorcells in lungs were quantified by means of immunohistology and imageanalysis. Data represent means±SEM; significant differences vs. salinetreated controls were not found.

FIG. 5: Effect of chronic intragastric infusion of isoleucylthiazolidine fumarate on body weight change in grams in F344 rats withlung metastasis. A dose dependent reduction of the loss of body weightafter chronic infusion of different dosages of isoleucyl thiazolidinefumarate in F344 rats 2 weeks after injection of MADB106 tumor cells isillustrated. One factor ANOVA revealed a significant effect on bodyweight, which became significant in the post-hoc analysis at the 0.4 mgand 4 mg dosages. Data represent means±SEM; *p<0.05 reflectingsignificant differences vs. saline treated SHAM controls determined byFisher PLSD.

FIG. 6: Effect of chronic intragastric infusion of isoleucylthiazolidine fumarate on the number of lung tumor colonies in F344 rats.A dose dependent reduction of lung colony numbers after chronic infusionof different dosages of isoleucyl thiazolidine fumarate in F344 rats 2weeks after injection of MADB106 tumor cells is illustrated. One factorANOVA revealed a significant effect, which became significant in thepost-hoc analysis at the 4 mg dosage. Data represent means±SEM; *p<0.05reflecting significant differences vs. saline treated SHAM controlsdetermined by Fisher PLSD.

FIG. 7: Effect of chronic intragastric infusion of isoleucylthiazolidine fumarate; isoleucyl cyanopyrrolidine TFA, and valylpyrrolidine fumarate on the number of lung tumor colonies in F344 rats.A significant reduction of lung colony numbers after chronic infusion ofisoleucyl thiazolidine fumarate and isoleucyl cyanopyrrolidine TFA inF344 rats 2 weeks after injection of MADB106 tumor cells is illustrated.Data represent means±SEM; *p<0.05 reflecting significant differences vs.saline treated SHAM controls determined by ANOVA and Fisher PLSD.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the area of dipeptidyl peptidase IV(DPIV) inhibition and, more particularly, to a new use of inhibitors ofDPIV and DPIV-like enzyme activity for the treatment of cancer andtumors, in particular for the prevention and inhibition of metastasisand tumor colonization, and pharmaceutical compositions containing saidcompounds.

In contrast to other proposed methods in the art, the present inventionprovides an orally available therapy with low molecular weightinhibitors of dipeptidyl peptidase IV. The instant invention representsa novel approach for the treatment of cancer and metastatic disease. Itis user friendly, commercially useful and suitable for use in atherapeutic regime, especially concerning human diseases.

Spontaneous mutations of the DPIV gene observed in substrains of F344rats provide a model for studying the role of DPIV in tumor adhesion andcolonization. The mutations in F344 rats result in a lack of DPIVenzymatic activity and are found in substrains from Germany (GER) andJapan (JAP) (Thompson et al, 1991; Tsuji et al, 1992), while rats fromUSA breeders show significant enzyme activity. In F344JAP rats, a G633Rsubstitution in the DPIV protein causes markedly reduced expression of amutant inactive enzyme (Cheng et al, 1999; Tsuji et al, 1992;), whilethe other DPIV negative F344GER substrain expresses a non-active mutantenzyme (Thompson et al, 1991). Studies by Pauli and co-workers(Abdel-Ghany et al, 1998; Cheng et al, 1998; Cheng et al, 1999; Johnsonet al, 1993) have demonstrated an important role of DPIV/Fibronectinbinding in lung metastasis and have discussed the F344JAP rat as a“protein knock-out” model, although this substrain expresses mutant,enzymatically inactive DPIV on endothelial cell surfaces, albeit atgreatly reduced levels when compared to expression of wild type DPIV(Cheng et al, 1999).

On the basis of these findings, the investigation of the role of DPIVexpression and enzymic activity in cancer and cancer according to thepresent invention revealed the oral administration of DPIV inhibitors inresults in a decrease of lung metastasis and colonization.

The goal of the present invention is the development of dipeptidylpeptidase IV inhibitors and/or ligands, which display a highbioavailability. In another preferred embodiment, the present inventionprovides DPIV inhibitors, which have an exactly predictable activitytime in the target tissue.

Examples for target specific, orally available low molecular weightagents are prodrugs of stable and unstable dipeptidyl peptidase IVinhibitors which comprise general formula A-B-C, whereby A represents anamino acid, B represents the chemical bond between A and C or an aminoacid, and C represents an unstable or a stable inhibitor of dipeptidylpeptidase IV respectively. They are described in WO 99/67278, WO99/67279 the teachings of which are herein incorporated by reference intheir entirety.

The present invention relates to a novel method, in which the reductionof activity in the enzyme dipeptidyl peptidase (DPIV or CD 26), or ofDPIV-like enzyme activity, or where binding of a DPIV specific ligandexerts tumor suppressive or immunostimulating effects in the organismsof mammals induced by effectors of the enzyme leads as a causalconsequence to a reduced growth or adhesion of cancer cells. Suchtreatment will result in a reduction or delay of cancer cell adhesion(metastasis) or the growth of tumor. As a consequence mammals bearingcancer will benefit from the treatment with inhibitors of DPIV aDPIV-like enzyme activity.

The method of the present invention for treating cancer in an animal,including humans, in need thereof, comprises anti-cancer effects bybinding or by inhibiting DPIV, or related enzyme activities, using aninhibitor or ligand of these enzymes. Oral administration of a DPIVinhibitor may be preferable in most circumstances.

The present invention will now be illustrated with reference to thefollowing examples focusing on the anti-cancer-like andanti-metastatic-like action of reduced DPIV-like activity and/or bindingin an in vivo cancer cell adhesion assay (example 13), and in cancercolonization assays (example 14).

In one illustrative embodiment, the present invention relates todipeptide compounds and compounds analogous to dipeptide compounds thatare formed from an amino acid and a thiazolidine or pyrrolidine group,and salts thereof, referred to hereinafter as dipeptide compounds.

The use of such compounds as inhibitors of DPIV or of DPIV-analogousenzyme activity is already known from DD 296 075, PCT/DE 97/00820 andPCT/EP 99/03712.

Especially suitable for that purpose according to the invention aredipeptide compounds in which the amino acid is selected from a naturalamino acid, such as, for example, leucine, valine, glutamine, glutamicacid, proline, isoleucine, asparagines and aspartic acid.

The dipeptide compounds according to the invention exhibit at aconcentration (of dipeptide compounds) of 10 μM, especially under theconditions indicated in Table 1, a reduction in the activity ofdipeptidyl peptidase IV or DPIV-analogous enzyme activities of at least10%, especially of at least 40%. Frequently a reduction in activity ofat least 60% or at least 70% is also required. Preferred effectors mayalso exhibit a reduction in activity of a maximum of 20% or 30%.

Preferred compounds are L-allo-isoleucyl thiazolidine, L-threo-isoleucylpyrrolidine and salts thereof, especially the fumaric salts, andL-allo-isoleucyl pyrrolidine and salts thereof. Especially preferredcompounds are glutaminyl pyrrolidine and glutaminyl thiazolidine offormulas 1 and 2:

Further preferred compounds are given in Table 1.The salts of the dipeptide compounds can be present in a molar ration ofdipeptide (-analogous) component to salt component of 1:1 or 2:1. Such asalt is, for example, (Ile-Thia)₂ fumaric acid.

TABLE 1 Structures of further preferred dipeptide compounds EffectorH-Asn-pyrrolidine H-Asn-thiazolidine H-Asp-pyrrolidineH-Asp-thiazolidine H-Asp(NHOH)-pyrrolidine H-Asp(NHOH)-thiazolidineH-Glu-pyrrolidine H-Glu-thiazolidine H-Glu(NHOH)-pyrrolidineH-Glu(NHOH)-thiazolidine H-His-pyrrolidine H-His-thiazolidineH-Pro-pyrrolidine H-Pro-thiazolidine H-Ile-azididine H-Ile-pyrrolidineH-L-allo-Ile-thiazolidine H-Val-pyrrolidine H-Val-thiazolidine

In another preferred embodiment, the present invention provides peptidecompounds of formula 3 useful for competitive modulation of dipeptidylpeptidase IV catalysis:

wherein

A, B, C, D and E are any amino acid residues including proteinogenicamino acids, non-proteinogenic amino acids, L-amino acids and D-aminoacids and wherein E and/or D may be absent or B and/or A may be absentwith additional conditions as hereinafter detailed:

-   -   Further conditions regarding formula (3):    -   A is any amino acid residue except D-amino acid residues;    -   B is any proteinogenic amino acid residue, but

If B is an amino acid selected from Pro, Ala, Ser, Gly, Hyp,acetidine-(2)-carboxylic acid or pipecolic acid, then C is any aminoacid residue including D-amino acids, except Pro, Ala, Ser, Gly, Hyp,acetidine-(2)-carboxylic acid or pipecolic acid and E may be unused togenerate tetrapeptides of the formula A-B-C-D, or D and E may be unusedto generate tripeptides of the formula A-B-C provided, but

If B is not an amino acid selected from Pro, Ala, Ser, Gly, Hyp,acetidine-(2)-carboxylic acid or pipecolic acid, then C is any α-aminoacid except D-amino acids; D is Pro, Ala, Ser, Gly, Hyp,acetidine-(2)-carboxylic acid or pipecolic acid; E is any amino acidresidue including D-amino acids, except Pro, Ala, Ser, Gly, Hyp,acetidine-(2)-carboxylic acid or pipecolic acid, but

If D is Pro, Ala, Ser, Gly, Hyp, acetidine-(2)-carboxylic acid orpipecolic acid, then C is any α-amino acid except D-amino acids and Pro,Ala, Ser, Gly, Hyp, acetidine-(2)-carboxylic acid or pipecolic acid, andA may be unused to generate tetrapeptides of the formula B-C-D-E, or Aand B may be unused to generate tripeptides of the formula C-D-Eprovided however,

If D is not selected from Pro, Ala, Ser, Gly, Hyp,acetidine-(2)-carboxylic acid or pipecolic acid, then E is any aminoacid residue including D-amino acids.

Amino acid residues used for the preparation of the compounds of formula(3) can be generally subclassified into four major subclasses asfollows.

-   Acidic: The residue has a negative charge due to loss of H ion at    physiological pH and the residue is attracted by aqueous solution so    as to seek the surface positions in the conformation of a peptide in    which it is contained when the peptide is in aqueous medium at    physiological pH.-   Basic: The residue has a positive charge due to association with H    ion at physiological pH and the residue is attracted by aqueous    solution so as to seek the surface positions in the conformation of    a peptide in which it is contained when the peptide is in aqueous    medium at physiological pH.-   Neutral/nonpolar: The residues are not charged at physiological pH    and the residue is repelled by aqueous solution so as to seek the    inner positions in the conformation of a peptide in which it is    contained when the peptide is in aqueous medium. These residues are    also designated “hydrophobic” herein.-   Neutral/polar: The residues are not charged at physiological pH, but    the residue is attracted by aqueous solution so as to seek the outer    positions in the conformation of a peptide in which it is contained    when the peptide is in aqueous medium.

It is understood, of course, that in a statistical collection ofindividual residue molecules some molecules will be charged, and somenot, and there will be an attraction for or repulsion from an aqueousmedium to a greater or lesser extent. To fit the definition of“charged”, a significant percentage (at least approximately 25%) of theindividual molecules are charged at physiological pH. The degree ofattraction or repulsion required for classification as polar or nonpolaris arbitrary, and, therefore, amino acids specifically contemplated bythe invention have been specifically classified as one or the other.Most amino acids not specifically named can be classified on the basisof known behavior.

Amino acid residues can be further subclassified as cyclic or noncyclic,and aromatic or nonaromatic, self-explanatory classifications withrespect to the side chain substituent groups of the residues, and assmall or large. The residue is considered small if it contains a totalof 4 carbon atoms or less, inclusive of the carboxyl carbon. Smallresidues are, of course, always nonaromatic.

For the naturally occurring protein amino acids, subclassificationaccording to the foregoing scheme is as follows.

-   Acidic: Aspartic acid and Glutamic acid;-   Basic/noncyclic: Arginine, Lysine;-   Basic/cyclic: Histidine;-   Neutral/polar/small: Glycine, Serine and Cysteine;-   Neutral/polar/large/nonaromatic: Threonine, Asparagine, Glutamine;-   Neutral/polar/large/aromatic: Tyrosine;-   Neutral/nonpolar/small: Alanine;-   Neutral/nonpolar/large/nonaromatic: Valine, Isoleucine, Leucine,    Methionine;-   Neutral/nonpolar/large/aromatic: Phenylalanine, and Tryptophan.

The gene-encoded amino acid proline, although technically within thegroup neutral/nonpolar/large/cyclic and nonaromatic, is a special casedue to its known effects on the secondary conformation of peptidechains, and is not, therefore, included in this specific defined group.

Certain commonly encountered amino acids, which are not encoded by thegenetic code, include, for example, beta-alanine (beta-ala), or otheromega-amino acids, such as 3-amino propionic, 4-amino butyric and soforth, alpha-aminoisobutyric acid (Aib), sarcosine (Sar), ornithine(Orn), citrulline (Cit), homoarginine (Har), t-butylalanine(t-butyl-Ala), t-butylglycine (t-butyl-Gly), N-methylisoleucine(N-Melle), phenylglycine (Phg), cyclohexylalanine (Cha), norleucine(Nle), cysteic acid (Cya) and methionine sulfoxide (MSO). These alsofall conveniently into particular categories.

-   -   Based on the above definition,    -   Sar and beta-Ala are neutral/nonpolar/small;    -   t-butyl-Ala, t-butyl-Gly, N-Melle, Nle and Cha are        neutral/nonpolar/large/nonaromatic;    -   Har and Orn are basic/noncyclic;    -   Cya is acidic;    -   Cit, Acetyl-Lys, and MSO are neutral/polar/large/nonaromatic;        and    -   Phg is neutral/nonpolar/large/aromatic.    -   The various omega-amino acids are classified according to size        as neutral/nonpolar/small (beta-Ala, i.e., 3-aminopropionic,        4-aminobutyric) or large (all others).

Other amino acid substitutions for those encoded in the genetic code canalso be included in peptide compounds within the scope of the inventionand can be classified within this general scheme.

Proteinogenic amino acids are defined as natural protein-derived α-aminoacids. Non-proteinogenic amino acids are defined as all other aminoacids, which are not building blocks of common natural proteins.

The resulting peptides may be synthesized as the free C-terminal acid oras the C-terminal amide form. The free acid peptides or the amides maybe varied by side chain modifications. Such side chain modificationsinclude for instance, but not restricted to, homoserine formation,pyroglutamic acid formation, disulphide bond formation, deamidation ofasparagine or glutamine residues, methylation, t-butylation,t-butyloxycarbonylation, 4-methylbenzylation, thioanysilation,thiocresylation, bencyloxymethylation, 4-nitrophenylation,bencyloxycarbonylation, 2-nitrobencoylation, 2-nitrosulphenylation,4-toluenesulphonylation, pentafluorophenylation, diphenylmethylation,2-chlorobenzyloxycarbonylation, 2,4,5-trichlorophenylation,2-bromobenzyloxycarbonylation, 9-fluorenylmethyloxycarbonylation,triphenylmethylation, 2,2,5,7,8,-pentamethylchroman-6-sulphonylation,hydroxylation, oxidation of methionine, formylation, acetylation,anisylation, bencylation, bencoylation, trifluoroacetylation,carboxylation of aspartic acid or glutamic acid, phosphorylation,sulphation, cysteinylation, glycolysation with pentoses, deoxyhexoses,hexosamines, hexoses or N-acetylhexosamines, farnesylation,myristolysation, biotinylation, palmitoylation, stearoylation,geranylgeranylation, glutathionylation, 5′-adenosylation,ADP-ribosylation, modification with N-glycolylneuraminic acid,N-acetylneuraminic acid, pyridoxal phosphate, lipoic acid,4′-phosphopantetheine, or N-hydroxysuccinimide.

In the compounds of formula (3), the amino acid residues comprising A,B, C, D, and E substituents are attached to the adjacent moietyaccording to standard nomenclature so that the amino-terminus(N-terminus) of the amino acids is drawn on the left and thecarboxyl-terminus of the amino acid is drawn to the right. Until thepresent invention by Applicants, known peptide substrates of theproline-specific serine protease dipeptidyl peptidase IV in vitro arethe tripeptides Diprotin A (IIe-Pro-IIe), Diprotin B (Val-Pro-Leu) andDiprotin C (Val-Pro-IIe). Applicants have unexpectedly discovered thatthe compounds disclosed here act as substrates of dipeptidyl peptidaseIV in vivo in a mammal and, in pharmacological doses, inhibit thephysiological turnover of endogenous substrates by competitivecatalysis.

Particularly preferred compounds of the present invention that could beuseful as modulators of dipeptidyl peptidase IV and DPIV—like enzymesinclude those compounds which show K_(i)-values for DPIV-binding,effectively in DPIV-inhibition in vivo after i.v. and/or p.o.administration to Wistar rats

Further preferred compounds according to the present invention arepeptidylketones of formula 4:

wherein

-   A is selected from:

-   X¹ is H or a acyl or oxycarbonyl group incl. all amino acids and    peptide residues,-   X² is H, —(CH)_(n)—NH—C₅H₃N—Y with n=2–4 or C₅H₃N—Y (a divalent    pyridyl residue) and Y is selected from H, Br, Cl, I, NO₂ or CN,    -   X³ is H or selected from an alkyl, alkoxy, halogen, nitro, cyano        or carboxy substituted phenyl or pyridyl residue,    -   X⁴ is H or selected from an alkyl, alkoxy, halogen, nitro, cyano        or carboxy substituted phenyl or pyridyl residue,    -   X⁵ is H or an alkyl, alkoxy or phenyl residue,    -   X⁶ is H or a alkyl residue.-   for n=1    -   X is selected from: H, OR², SR², NR²R³, N⁺R²R³R⁴, wherein:    -   R² stands for acyl residues, which are substituted with alkyl,        cycloalkyl, aryl or heteroaryl residues, or for all amino acids        and peptidic residues, or alkyl residues, which are substituted        with alkyl, cycloalkyl, aryl and heteroaryl residues,    -   R³ stands for alkyl and acyl functions, wherein R² and R³ may be        embedded in ring structures of saturated and unsaturated        carbocyclic or heterocyclic structures,    -   R⁴ stands for alkyl residues, wherein R² and R⁴ or R³ and R⁴ may        be embedded in ring structures of saturated and unsaturated        carbocyclic or heterocyclic structures.-   for n=0    -   X is selected from:

-   -    wherein

-   B stands for: O, S, NR⁵, wherein R⁵ is H, a alkyl or acyl,    -   C, D, E, F, G, H are independently selected from alkyl and        substituted alkyl residues, oxyalkyl, thioalkyl, aminoalkyl,        carbonylalkyl, acyl, carbamoyl, aryl and heteroaryl residues;        and

-   Z is selected from H, or a branched or single chain alkyl residue    from C₁–C₉ or a branched or single chain alkenyl residue from C₂–C₉,    a cycloalkyl residue from C₃–C₈, a cycloalkenyl residue from C₅–C₇,    a aryl- or heteroaryl residue, or a side chain selected from all    side chains of all natural amino acids or derivatives thereof.    Further, the present invention provides compounds of formulas 5, 6,    7, 8, 9, 10 and 11, including all stereoisomers and pharmaceutical    acceptable salts thereof,

wherein:

-   R¹ is H, a branched or linear C₁–C₉ alkyl residue, a branched or    linear C₂–C₉ alkenyl residue, a C₃–C₈ cycloalkyl-, C₅–C₇    cycloalkenyl-, aryl- or heteroaryl residue or a side chain of a    natural amino acid or a derivative thereof,-   R³ and R⁴ are selected from H, hydroxy, alkyl, alkoxy, aryloxy,    nitro, cyano or halogen,-   A is H or an isoster of an carbonic acid, like a functional group    selected from CN, SO₃H, CONHOH, PO₃R⁵R⁶, tetrazole, amide, ester,    anhydride, thiazole and imidazole,-   B is selected from:

-    wherein:-   R⁵ is H, —(CH)_(n)—NH—C₅H₃N—Y with n=2–4 and C₅H₃N—Y (a divalent    pyridyl residue) with Y=H, Br, Cl, I, NO₂ CN,-   R¹⁰ is H, a acyl, oxycarbonyl or a amino acid residue,-   W is H or a phenyl or pyridyl residue, substituted with alkyl,    alkoxy, halogen, nitro, cyano or carboxy residue,-   W¹ is H, a alkyl, alkoxy or phenyl residue,-   Z is H or a phenyl or pyridyl residue, substituted with alkyl,    alkoxy, halogen, nitro, cyano or carboxy residue,-   Z¹ is H or a alkyl residue,-   D is a cyclic C₄–C₇ alkyl, C₄–C₇ alkenyl residue or a alkyl    substituted derivative thereof or a cyclic 4–7-membered heteroalkyl    or 4–7-membered heteroalkenyl residue,-   X² is O, NR⁶, N⁺(R⁷)₂, or S,-   X³ to X¹² are selected from CH₂, CR⁸R⁹, NR⁶, N⁺(R⁷)₂, O, S, SO and    SO₂, O, S, SO and SO₂, including all saturated and unsaturated    structures,-   R⁶, R⁷, R⁸, R⁹ are selected from H, a branched or linear C₁–C₉ alkyl    residue, a branched or linear C₂–C₉ alkenyl residue, a C₃–C₈    cycloalkyl residue, a C₅–C₇ cycloalkenyl residue, an aryl or    heteroaryl residue,-   with the following provisions:-   Formula 6: X⁶ is CH if A is not H,-   Formula 7: X¹⁰ is C if A is not H,-   Formula 8: X⁷ is CH if A is not H,-   Formula 9: X¹² is C if A is not H.

Because of the wide distribution of the protein in the body and the widevariety of mechanisms involving DPIV, DPIV-activity and DPIV-relatedproteins, systemic therapy (enteral or parenteral administration) withDPIV-inhibitors can result in a series of undesirable side-effects.

It has been possible to show that side chain-modified substrates of theenzyme dipeptidyl peptidase IV can be recognised by the enzyme andcleaved in the same way as unmodified substrates (DEMUTH, H.-U., HEINS,J., 1995).

For example, it has been possible to show that phosphorylateddipeptide-(B)-p-nitroanilides [KASPARI, A., et al., 1996] are substratesof DPIV. DPIV-inhibitors such as, for example, Glu(Gly)-Thia orLys(Z-NO₂)-Thia [REINHOLD, D., et al., 1998] are transported completely.

The problem to be solved consisted in preparing compounds that can beused for targeted influencing of locally limited pathophysiological andphysiological processes. The problem of the invention especiallyconsists in obtaining locally limited inhibition of DPIV orDPIV-analogous activity for the purpose of targeted intervention in theregulation of the activity of locally active substrates.

This problem is solved according to the invention by providing compoundsof the general formula (12)

wherein

-   A is an amino acid having at least one functional group in the side    chain,-   B is a chemical compound covalently bound to at least one functional    group of the side chain of A, namely    -   oligopeptides having a chain length of up to 20 amino acids,        except for homopolymers of glycine consisting of up to 6 glycine        monomers, or    -   polyethylene glycols having molar masses of up to 20 000 g/mol,        and-   C is a thiazolidine, pyrrolidine, cyanopyrrolidine, hydroxyproline,    dehydroproline or piperidine group amide-bonded to A.

In accordance with the invention, pharmaceutical compositions areprovided comprising at least one compound of the general formula (12)

wherein

-   A is an amino acid, preferably an α-amino acid, especially a natural    α-amino acid having at least one functional group in the side chain,    preferably threonine, tyrosine, serine, arginine, lysine, aspartic    acid, glutamic acid or cysteine,-   B is a chemical compound covalently bound to at least one functional    group in the side chain of A, namely oligopeptides having a chain    length of up to 20 amino acids, polyethylene glycols having molar    masses of up to 20 000 g/mol, optionally substituted organic amines,    amides, alcohols, acids or aromatic compounds having from 8 to 50 C    atoms,-   C is a thiazolidine, pyrrolidine, cyanopyrrolidine, hydroxyproline,    dehydroproline or piperidine group amide-bonded to A,    and at least one customary adjuvant appropriate for the site of    action.

Throughout the description and the claims for the compounds of formula(12), the expression “alkyl” can denote a C₁₋₅₀ alkyl group, preferablya C₆₋₃₀ alkyl group, especially a C₈₋₁₂ alkyl group; for example, analkyl group may be a methyl, ethyl, propyl, isopropyl or butyl group.The expression “alk”, for example in the expression “alkoxy”, and theexpression “alkan”, for example in the expression “alkanoyl”, aredefined as for “alkyl”; aromatic compounds are preferably substituted oroptionally unsubstituted phenyl, benzyl, naphthyl, biphenyl oranthracene groups, which preferably have at least 8 C atoms; theexpression “alkenyl” can denote a C₂₋₁₀ alkenyl group, preferably a C₂₋₆alkenyl group, which has the double bond(s) at any desired location andmay be substituted or unsubstituted; the expression “alkynyl” can denotea C₂₋₁₀ alkynyl group, preferably a C₂₋₆ alkynyl group, which has thetriple bond(s) at any desired location and may be substituted orunsubstituted; the expression “substituted” or substituent can denoteany desired substitution by one or more, preferably one or two, alkyl,alkenyl, alkynyl, mono- or multi-valent acyl, alkanoyl, alkoxyalkanoylor alkoxyalkyl groups; the afore-mentioned substituents may in turn haveone or more (but preferably zero) alkyl, alkenyl, alkynyl, mono- ormulti-valent acyl, alkanoyl, alkoxyalkanoyl or alkoxyalkyl groups asside groups; organic amines, amides, alcohols or acids, each having from8 to 50 C atoms, preferably from 10 to 20 C atoms, can have the formulae(alkyl)₂N— or alkyl-NH—, —CO—N(alkyl)₂ or —CO—NH(alkyl), -alkyl-OH or-alkyl-COOH.

Despite an extended side chain function, the compounds of formula (12)can still bind to the active centre of the enzyme dipeptidyl peptidaseIV and analogous enzymes but are no longer actively transported by thepeptide transporter PepT1. The resulting reduced or greatly restrictedtransportability of the compounds according to the invention leads, inideal manner, to local or site directed inhibition of DPIV and DPIV-likeenzyme activity.

The compounds of formula (12) or compounds used in accordance with theinvention can be present or used, respectively, in the form of racematesor in the form of enantiomerically pure compounds, preferably in theL-threo or L-allo form with respect to part A of formula (12).

By extending/expanding the side chain modifications, for example beyonda number of seven carbon atoms, it is accordingly possible to obtain adramatic reduction in transportability (see Example 12). The Examples inTable 12.1 clearly show that, with increasing spatial size of the sidechains, there is a reduction in the transportability of the substances.By spatially and sterically expanding the side chains, for examplebeyond the atom group size of a monosubstituted phenyl radical,hydroxylamine radical or amino acid residue, it is possible according tothe invention to modify or suppress the transportability of the targetsubstances.

According to the present invention, the compounds of formula (12)inhibit DPIV or DPIV-like enzyme activity in the body of a mammal in asite specific manner. It is accordingly possible to influence localphysiological and pathophysiological conditions (inflammation,psoriasis, arthritis, autoimmune diseases, allergies) effectively andwith dramatically reduced side-effects.

Preferred compounds of formula (12) are compounds, wherein theoligopeptides have chain lengths of from 3 to 15, especially from 4 to10, amino acids, and/or the polyethylene glycols have molar masses of atleast 250 g/mol, preferably of at least 1500 g/mol and up to 15 000g/mol, and/or the optionally substituted organic amines, amides,alcohols, acids or aromatic compounds have at least 12 C atoms andpreferably up to 30 C atoms.

The compounds of the present invention can be converted into acidaddition salts, especially pharmaceutically acceptable acid additionsalts. The pharmaceutically acceptable salt generally takes a form inwhich an amino acids basic side chain is protonated with an inorganic ororganic acid. Representative organic or inorganic acids includehydrochloric, hydrobromic, perchloric, sulfuric, nitric, phosphoric,acetic, propionic, glycolic, lactic, succinic, maleic, fumaric, malic,tartaric, citric, benzoic, mandelic, methanesulfonic,hydroxyethanesulfonic, benzenesulfonic, oxalic, pamoic,2-naphthalenesulfonic, p-toulenesulfonic, cyclohexanesulfamic,salicylic, saccharinic or trifluoroacetic acid. All pharmaceuticallyacceptable acid addition salt forms of the compounds of formulas (1) to(12) are intended to be embraced by the scope of this invention.

In view of the close relationship between the free compounds and thecompounds in the form of their salts, whenever a compound is referred toin this context, a corresponding salt is also intended, provided such ispossible or appropriate under the circumstances.

The present invention further includes within its scope prodrugs of thecompounds of this invention. In general, such prodrugs will befunctional derivatives of the compounds which are readily convertible invivo into the desired therapeutically active compound. Thus, in thesecases, the methods of treatment of the present invention, the term“administering” shall encompass the treatment of the various disordersdescribed with prodrug versions of one or more of the claimed compounds,but which converts to the above specified compound in vivo afteradministration to the subject. Conventional procedures for the selectionand preparation of suitable prodrug derivatives are described, forexample, in “Design of Prodrugs”, ed. H. Bundgaard, Elsevier, 1985 andthe patent applications DE 198 28 113 and DE 198 28 114, which are fullyincorporated herein by reference.

Where the compounds according to this invention have at least one chiralcenter, they may accordingly exist as enantiomers. Where the compoundspossess two or more chiral centers, they may additionally exist asdiastereomers. It is to be understood that all such isomers and mixturesthereof are encompassed within the scope of the present invention.Furthermore, some of the crystalline forms of the compounds may exist aspolymorphs and as such are intended to be included in the presentinvention. In addition, some of the compounds may form solvates withwater (i.e. hydrates) or common organic solvents, and such solvates arealso intended to be encompassed within the scope of this invention.

The compounds, including their salts, can also be obtained in the formof their hydrates, or include other solvents used for theircrystallization.

As indicated above, the compounds of the present invention, and theircorresponding pharmaceutically acceptable acid addition salt forms, areuseful in inhibiting DPIV and DPIV—like enzyme activity. The ability ofthe compounds of the present invention, and their correspondingpharmaceutically acceptable acid addition salt forms to inhibit DPIV andDPIV—like enzyme activity may be demonstrated employing the DPIVactivity assay for determination of the K_(i)-values and the IC₅₀-valuesin vitro, as described in examples 7 and 8.

The ability of the compounds of the present invention, and theircorresponding pharmaceutically acceptable acid addition salt forms toinhibit DPIV in vivo may be demonstrated by oral or intravasaladministration to Wistar rats, as described in example 11. The compoundsof the present invention inhibit DPIV activity in vivo after both, oraland intravasal administration to Wistar rats.

DPIV is present in a wide variety of mammalian organs and tissues e.g.the intestinal brush-border (Gutschmidt S. et al., “Insitu”—measurements of protein contents in the brush border region alongrat jejunal villi and their correlations with four enzyme activities.Histochemistry 1981, 72 (3), 467–79), exocrine epithelia, hepatocytes,renal tubuli, endothelia, myofibroblasts (Feller A. C. et al., Amonoclonal antibody detecting dipeptidylpeptidase IV in human tissue.Virchows Arch. A. Pathol. Anat. Histopathol. 1986; 409 (2):263–73),nerve cells, lateral membranes of certain surface epithelia, e.g.Fallopian tube, uterus and vesicular gland, in the luminal cytoplasm ofe.g., vesicular gland epithelium, and in mucous cells of Brunner's gland(Hartel S. et al., Dipeptidyl peptidase (DPP) IV in rat organs.Comparison of immunohistochemistry and activity histochemistry.Histochemistry 1988; 89 (2): 151–61), reproductive organs, e.g. caudaepididymis and ampulla, seminal vesicles and their secretions (Agrawal &Vanha-Perttula, Dipeptidyl peptidases in bovine reproductive organs andsecretions. Int. J. Androl. 1986, 9 (6): 435–52). In human serum, twomolecular forms of dipeptidyl peptidase are present (Krepela E. et al.,Demonstration of two molecular forms of dipeptidyl peptidase IV innormal human serum. Physiol. Bohemoslov. 1983, 32 (6): 486–96). Theserum high molecular weight form of DPIV is expressed on the surface ofactivated T cells (Duke-Cohan J. S. et al., Serum high molecular weightdipeptidyl peptidase IV (CD26) is similar to a novel antigen DPPT-Lreleased from activated T cells. J. Immunol. 1996, 156 (5): 1714–21).

The compounds of the present invention, and their correspondingpharmaceutically acceptable acid addition salt forms are able to inhibitDPIV in vivo. In one embodiment of the present invention, all molecularforms, homologues and epitopes of DPIV from all mammalian tissues andorgans, also of those, which are undiscovered yet, are intended to beembraced by the scope of this invention.

Among the rare group of proline-specific proteases, DPIV was originallybelieved to be the only membrane-bound enzyme specific for proline asthe penultimate residue at the amino-terminus of the polypeptide chain.However, other molecules, even structurally non-homologous with the DPIVbut bearing corresponding enzyme activity, have been identifiedrecently. DPIV-like enzymes, which are identified so far, are e.g.fibroblast activation protein α, dipeptidyl peptidase IV β, dipeptidylaminopeptidase-like protein, N-acetylated α-linked acidic dipeptidase,quiescent cell proline dipeptidase, dipeptidyl peptidase II, attractinand dipeptidyl peptidase IV related protein (DPP 8), and are describedin the review article by Sedo & Malik (Sedo & Malik, Dipeptidylpeptidase IV-like molecules: homologous proteins or homologousactivities? Biochimica et Biophysica Acta 2001, 36506: 1–10). FurtherDPIV like enzymes are disclosed in WO 01/19866, WO 02/04610 and WO02/34900. WO 01/19866 discloses novel human dipeptidyl aminopeptidase(DPP8) with structural und functional similarities to DPIV andfibroblast activation protein (FAP). The dipeptidyl peptidase IV-likeenzyme of WO 02/04610 is well known in the art. In the Gene Bank database, this enzyme is registered as KIAA1492. In another preferredembodiment of the present invention, all molecular forms, homologues andepitopes of proteins comprising DPIV-like enzyme activity, from allmammalian tissues and organs, also of those, which are undiscovered yet,are intended to be embraced by the scope of this invention.

The ability of the compounds of the present invention, and theircorresponding pharmaceutically acceptable acid addition salt forms toinhibit DPIV-like enzymes may be demonstrated employing an enzymeactivity assay for determination of the K_(i)-values in vitro asdescribed in example 9. The K_(i)-values of the compounds of the presentinvention against porcine dipeptidyl peptidase II were exemplarydetermined as K_(i)=8.52*10⁻⁵ M±6.33*10⁻⁶ M for glutaminyl pyrrolidineand K_(i)=1.07*10⁻⁵ M±3.81*10⁻⁷ M for glutaminyl thiazolidine.

In another embodiment, the compounds of the present invention, and theircorresponding pharmaceutically acceptable acid addition salt forms haveonly low, if no inhibitory activity against non-DPIV and non-DPIV-likeproline specific enzymes. As described in example 10, with glutaminylthiazolidine and glutaminyl pyrrolidine exemplarily, no inhibition ofdipeptidyl peptidase I and prolyl oligopeptidase was found. Againstprolidase, both compounds explained a marked lower efficacy compared toDPIV. The IC 50-values against prolidase were determined as IC 50>3 mMfor glutaminyl thiazolidine and as IC 50=3.4*10⁻⁴M±5.63*10⁻⁵ forglutaminyl pyrrolidine.

The present invention provides a method of treating a condition mediatedby modulation of the DPIV or DPIV-like enzyme activity in a subject inneed thereof which comprises administering any of the compounds of thepresent invention or pharmaceutical compositions thereof in a quantityand dosing regimen therapeutically effective to treat the condition.Additionally, the present invention includes the use of the compounds ofthis invention, and their corresponding pharmaceutically acceptable acidaddition salt forms, for the preparation of a medicament for thetreatment of a condition mediated by modulation of the DPIV activity ina subject. The compound may be administered to a patient by anyconventional route of administration, including, but not limited to,intravenous, oral, subcutaneous, intramuscular, intradermal andparenteral.

In a further illustrative embodiment, the present invention providesformulations for the compounds of formulas 1 to 12, and theircorresponding pharmaceutically acceptable acid addition salt forms, inpharmaceutical compositions.

The term “subject” as used herein, refers to an animal, preferably amammal, most preferably a human, who has been the object of treatment,observation or experiment.

The term “therapeutically effective amount” as used herein, means thatamount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue system, animal or human,being sought by a researcher, veterinarian, medical doctor or otherclinician, which includes alleviation of the symptoms of the disease ordisorder being treated.

As used herein, the term “composition” is intended to encompass aproduct comprising the claimed compounds in the therapeuticallyeffective amounts, as well as any product which results, directly orindirectly, from combinations of the claimed compounds.

To prepare the pharmaceutical compositions of this invention, one ormore compounds of formulas 1 to 12, and their correspondingpharmaceutically acceptable acid addition salt forms, as the activeingredient, is intimately admixed with a pharmaceutical carrieraccording to conventional pharmaceutical compounding techniques, whichcarrier may take a wide variety of forms depending of the form ofpreparation desired for administration, e.g., oral or parenteral such asintramuscular. In preparing the compositions in oral dosage form, any ofthe usual pharmaceutical media may be employed. Thus, for liquid oralpreparations, such as for example, suspensions, elixirs and solutions,suitable carriers and additives may advantageously include water,glycols, oils, alcohols, flavoring agents, preservatives, coloringagents and the like; for solid oral preparations such as, for example,powders, capsules, gelcaps and tablets, suitable carriers and additivesinclude starches, sugars, diluents, granulating agents, lubricants,binders, disintegrating agents and the like. Because of their ease inadministration, tablets and capsules represent the most advantageousoral dosage unit form, in which case solid pharmaceutical carriers areemployed. If desired, tablets may be sugar coated or enteric coated bystandard techniques. For parenterals the carrier will usually comprisesterile water, through other ingredients, for example, for purposes suchas aiding solubility or for preservation, may be included.

Injectable suspensions may also prepared, in which case appropriateliquid carriers, suspending agents and the like may be employed. Thepharmaceutical compositions herein will contain, per dosage unit, e.g.,tablet, capsule, powder, injection, teaspoonful and the like, an amountof the active ingredient necessary to deliver an effective dose asdescribed above. The pharmaceutical compositions herein will contain,per unit a dosage unit, e.g., tablet, capsule, powder, injection,suppository, teaspoonful and the like, of from about 0.03 mg to 100mg/kg (preferably 0.1–30 mg/kg) and may be given at a dosage of fromabout 0.1–300 mg/kg per day (preferably 1–50 mg/kg per day). Thedosages, however, may be varied depending upon the requirement of thepatients, the severity of the condition being treated and the compoundbeing employed. The use of either daily administration or post-periodicdosing may be employed. Typically the dosage will be regulated by thephysician based on the characteristics of the patient, his/her conditionand the therapeutic effect desired.

Preferably these compositions are in unit dosage forms from such astablets, pills, capsules, powders, granules, sterile parenteralsolutions or suspensions, metered aerosol or liquid sprays, drops,ampoules, autoinjector devices or suppositories; for oral parenteral,intranasal, sublingual or rectal administration, or for administrationby inhalation or insufflation. Alternatively, the composition may bepresented in a form suitable for once-weekly or once-monthlyadministration; for example, an insoluble salt of the active compound,such as the decanoate salt, may be adapted to provide a depotpreparation for intramuscular injection. For preparing solidcompositions such as tablets, the principal active ingredient is ideallymixed with a pharmaceutical carrier, e.g. conventional tabletingingredients such as corn starch, lactose, sucrose, sorbitol, talc,stearic acid, magnesium stearate, dicalcium phosphate or gums, and otherpharmaceutical diluents, e.g. water, to form a solid preformulationcomposition containing a homogeneous mixture of a compound of thepresent invention, or a pharmaceutically acceptable salt thereof. Whenreferring to these preformulation compositions as homogeneous, it ismeant that the active ingredient is ideally dispersed evenly throughoutthe composition so that the composition may be readily subdivided intoequally effective dosage forms such as tablets, pills and capsules. Thissolid preformulation composition may then be subdivided into unit dosageforms of the type described above containing from 0.1 to about 500 mg ofthe active ingredient of the present invention.

The tablets or pills of the novel composition can be advantageouslycoated or otherwise compounded to provide a dosage form affording theadvantage of prolonged action. For example, the tablet or pill cancomprise an inner dosage and an outer dosage component, the latter beingin the form of an envelope over the former. The two components can beseparated by an enteric layer which serves to resist disintegration inthe stomach and permits the inner component to pass intact into theduodenum or to be delayed in release. A variety of materials can be usedfor such enteric layers or coatings, such materials including a numberof polymeric acids with such materials as shellac, cetyl alcohol andcellulose acetate.

This liquid forms in which the novel compositions of the presentinvention may be advantageously incorporated for administration orallyor by injection include, aqueous solutions, suitably flavored syrups,aqueous or oil suspensions, and flavored emulsions with edible oils suchas cottonseed oil, sesame oil, coconut oil or peanut oil, as well aselixirs and similar pharmaceutical vehicles. Suitable dispersing orsuspending agents for aqueous suspensions include synthetic and naturalgums such as tragacanth, acacia, alginate, dextran, sodiumcarboxymethylcellulose, methylcellulose, polyvinylpyrrolidone orgelatin.

Where the processes for the preparation of the compounds according tothe invention give rise to mixture of stereoisomers, these isomers maybe separated by conventional techniques such as preparativechromatography. The compounds may be prepared in racemic form, orindividual enantiomers may be prepared either by enantiospecificsynthesis or by resolution. The compounds may, for example, be resolvedinto their components enantiomers by standard techniques, such as theformation of diastereomeric pairs by salt formation with an opticallyactive acid, such as (−)-di-p-toluoyl-d-tartaric acid and/or(+)-di-p-toluoyl-l-tartaric acid followed by fractional crystallizationand regeneration of the free base. The compounds may also resolved byformation of diastereomeric esters or amides, followed bychromatographic separation and removal of the chiral auxiliary.Alternatively, the compounds may be resolved using a chiral HPLC column.

During any of the processes for preparation of the compounds of thepresent invention, it may be necessary and/or desirable to protectsensitive or reactive groups on any of the molecules concerned. This maybe achieved by means of conventional protecting groups, such as thosedescribed in Protective Groups in Organic Chemistry, ed. J. F. W.McOmie, Plenum Press, 1973; and T. W. Greene & P. G. M. Wuts, ProtectiveGroups in Organic Synthesis, John Wiley & Sons, 1991, fully incorporatedherein by reference. The protecting groups may be removed at aconvenient subsequent stage using methods known from the art.

The method of treating conditions modulated by dipeptidyl peptidase IVand DPIV-like enzymes described in the present invention may also becarried out using a pharmaceutical composition comprising any of thecompounds as defined herein and a pharmaceutically acceptable carrier.The pharmaceutical composition may contain between about 0.01 mg and1000 mg, preferably about 5 to 500 mg, of the compound, and may beconstituted into any form suitable for the mode of administrationselected. Carriers include necessary and inert pharmaceuticalexcipients, including, but not limited to, binders, suspending agents,lubricants, flavorants, sweeteners, preservatives, dyes, and coatings.Compositions suitable for oral administration include solid forms, suchas pills, tablets, caplets, capsules (each including immediate release,timed release and sustained release formulations), granules, andpowders, and liquid forms, such as solutions, syrups, elixirs,emulsions, and suspensions. Forms useful for parenteral administrationinclude sterile solutions, emulsions and suspensions.

Advantageously, compounds of the present invention may be administeredin a single daily dose, or the total daily dosage may be administered individed doses of two, three or four times daily. Furthermore, compoundsof the present invention can be administered in intranasal form viatopical use of suitable intranasal vehicles, or via transdermal skinpatches well known to those of ordinary skill in that art. To beadministered in the form of transdermal delivery system, the dosageadministration will, of course, be continuous rather than intermittentthroughout the dosage regimen and dosage strength will need to beaccordingly modified to obtain the desired therapeutic effects.

More preferably, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic pharmaceutically acceptable inert carrier such as ethanol,glycerol, water and the like. Moreover, when desired or necessary,suitable binders; lubricants, disintegrating agents and coloring agentscan also be incorporated into the mixture. Suitable binders include,without limitation, starch, gelatin, natural sugars such as glucose orbetalactose, corn sweeteners, natural and synthetic gums such as acacia,tragacanth or sodium oleate, sodium stearate, magnesium stearate, sodiumbenzoate, sodium acetate, sodium chloride and the like. Disintegratorsinclude, without limitation, starch, methyl cellulose, agar, bentonite,xanthan gum and other compounds known within the art.

The liquid forms are suitable in flavored suspending or dispersingagents such as the synthetic and natural gums, for example, tragacanth,acacia, methyl-cellulose and the like. For parenteral administration,sterile suspensions and solutions are desired. Isotonic preparationswhich generally contain suitable preservatives are employed whenintravenous administration is desired.

The compound of the present invention can also be administered in theform of liposome delivery systems, such as small unilamellar vesicles,large unilamellar vesicles, and multilamellar vesicles. Liposomes can beformed from a variety of phospholipids, such as cholesterol,stearylamine or phosphatidylcholines using processes well described inthe art.

Compounds of the present invention may also be coupled with solublepolymers as targetable drug carriers. Such polymers can includepolyvinylpyrrolidone, pyran copolymer,polyhydroxypropylmethacrylamidephenol,polyhydroxyethylaspartamidephenol, or polyethyl eneoxidepolyllysinesubstituted with palmitoyl residue. Furthermore, compounds of thepresent invention may be coupled to a class of biodegradable polymersuseful in achieving controlled release of a drug, for example, polyacticacid, polyepsilon caprolactone, polyhydroxy butyeric acid,polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates andcross-linked or amphipathic block copolymers of hydrogels.

Compounds of this invention may be administered in any of the foregoingcompositions and according to dosage regimens established in the artwhenever treatment of the addressed disorders is required.

The daily dosage of the products may be varied over a wide range from0.01 to 1.000 mg per adult human per day. For oral administration, thecompositions are preferably provided in the form of tablets containing,0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 150,200, 250 and 500 milligrams of the active ingredient for the symptomaticadjustment of the dosage to the patient to be treated. An effectiveamount of the drug is ordinarily supplied at a dosage level of fromabout 0.1 mg/kg to about 300 mg/kg of body weight per day. Preferably,the range is from about 1 to about 50 mg/kg of body weight per day. Thecompounds may be administered on a regimen of 1 to 4 times per day.

Optimal dosages to be administered may be readily determined by thoseskilled in the art, and will vary with the particular compound used, themode of administration, the strength of the preparation, bioavailabilitydue to the mode of administration, and the advancement of diseasecondition. In addition, factors associated with the particular patientbeing treated, including patient age, weight, diet and time ofadministration, should generally be considered in adjusting dosages.

EXAMPLES Example 1 Synthesis of Dipeptide Compounds

1.1 General Synthesis of Isoleucyl Thiazolidine Salt

The Boc-protected amino acid BOC-Ile-OH is placed in ethyl acetate andthe batch is cooled to about −5° C. N-Methylmorpholine is addeddropwise, pivalic acid chloride (on a laboratory scale) or neohexanoylchloride (on a pilot-plant scale) is added dropwise at constanttemperature. The reaction is stirred for a few minutes for activation.N-Methylmorpholine (laboratory scale) and thiazolidine hydrochloride(laboratory scale) are added dropwise in succession, thiazolidine(pilot-plant scale) is added. Working-up in the laboratory is effectedin conventional manner using salt solutions, on a pilot-plant scale thebatch is purified with NaOH and CH₃COOH solutions.

The removal of the BOC protecting group is carried out using HCl/dioxane(laboratory scale) or H₂SO₄ (pilot-plant scale). In the laboratory thehydrochloride is crystallised from EtOH/ether.

On a pilot-plant scale the free amine is prepared by the addition ofNaOH/NH₃. Fumaric acid is dissolved in hot ethanol, the free amine isadded dropwise, and (IIe-Thia)² furmarate (M=520.71 gmol³¹ ¹)precipitates. The analysis of isomers and enantiomers is carried out byelectrophoresis.

1.2 Synthesis of Glutaminyl Pyrrolidine Free Base

Acylation:

N-Benzyl-oxycarbonylglutamine (2.02 g, 7.21 mmol) was dissolved in 35 mlTHF and brought to −15° C. Into that mixture CAIBE(isobutylchloroformiate) (0.937 ml, 7.21 mmol) and 4-methylmorpholine(0.795 ml, 7.21 mmol) where added and the solution was stirred for 15min. The formation of the mixed anhydride was checked by TLC (eluent:CHCl₃/MeOH: 9/1). After warming to −10° C. pyrrolidine (0.596 ml, 7.21mmol) was added. The mixture was brought to room temperature and stirredovernight.

Workup:

The sediment formed was filtered off and the solvent was evaporated. Theresulting oil was taken up in ethylacetate (20 ml) and washed with asaturated solution of sodiumhydrogensulfate followed by a saturatedsolution of sodiumbicarbonate, water and brine. The organic layer wasseparated, dried and evaporated. The resulting product was checked forpurity by TLC (eluent: CHCl₃/MeOH: 9/1).

Yield: 1.18 g, waxy solid.

Cleavage:

1.18 g of the resulting solid Z-protected compound was dissolved in 40ml absolute ethanol. Into the solution ca. 20 mg Pd on charcoal (10%,FLUKA) was added and the suspension was shaken under a hydrogenatmosphere for 3 h. The progress of the reaction was monitored by TLC(eluent: CHCl₃/MeOH: 9/1). After completion of the reaction the wasremoved to provide the free base.

Yield: 99%. The purity was checked by means of TLC:n-butanole/AcOH/water/ethylacetate: 1/1/1/1, R_(f)=0.4. The identity ofthe reaction product was checked by NMR analysis.

1.3 Synthesis of Glutaminyl Thiazolidine Hydrochloride

Acylation:

N-t-Butyl-oxycarbonylglutamine (2.0 g, 8.12 mmol) was dissolved in 5 mlTHF and brought to −15° C. Into that mixture CAIBE(isobutylchloroformiate) (1.06 ml, 8.12 mmol) and 4-methylmorpholine(0.895 ml, 8.12 mmol) where added and the solution was stirred for 15min. The formation of the mixed anhydride was checked by TLC (eluent:CHCl₃/MeOH: 9/1). After warming to −10° C. another equivalent4-methylmorpholine (0.895 ml, 8.12 mmol) and thiazolidinehydrochloride(1.02 g, 8.12 mmol was added. The mixture was brought to roomtemperature and stirred overnight.

Workup:

The sediment formed was filtered off and the solvent was evaporated. Theresulting oil was taken up in chloroform (20 ml) and washed with asaturated solution of sodiumhydrogensulfate followed by a saturatedsolution of sodiumbicarbonate, water and brine. The organic layer wasseparated, dried and evaporated. The resulting product was checked forpurity by TLC (eluent: CHCl₃/MeOH: 9/1).

Yield: 1.64 g, solid.

Cleavage:

640 mg of the resulting solid Boc-protected compound was dissolved in3.1 ml ice cold HCl in dioxane (12.98 M, 20 equivalents) and left onice. The progress of the reaction was monitored by TLC (eluent:CHCl₃/MeOH: 9/1). After completion of the reaction the solvent wasremoved and the resulting oil was taken up in methanole and evaporatedagain. After that the resulting oil was dried over phosphorous-V-oxideand triturated two times with diethylether. The purity was checked byHPLC.

Yield: 0.265 g. The purity was checked by HPLC. The identity of thereaction product was checked by NMR analysis.

1.4 Synthesis of Glutaminyl Pyrrolidine Hydrochloride

Acylation:

N-t-Butyl-oxycarbonylglutamine (3.0 g, 12.18 mmol) was dissolved in 7 mlTHF and brought to −15° C. Into that mixture CAIBE(isobutylchloroformiate) (1.6 ml, 12.18 mmol) and 4-methylmorpholine(1.3 ml, 12.18 mmol) where added and the solution was stirred for 15min. The formation of the mixed anhydride was checked by TLC (eluent:CHCl₃/MeOH: 9/1). After warming to −10° C. 1 equivalent of pyrrolidine(1.0 ml, 12.18 mmol) was added. The mixture was brought to roomtemperature and stirred overnight.

Workup:

The sediment formed was filtered off and the solvent was evaporated. Theresulting oil was taken up in chloroform (20 ml) and washed with asaturated solution of sodiumhydrogensulfate followed by a saturatedsolution of sodiumbicarbonate, water and brine. The organic layer wasseparated, dried and evaporated. The resulting product was checked forpurity by TLC (eluent: CHCl₃/MeOH: 9/1).

Yield: 2.7 g solid.

Cleavage:

2.7 g of the resulting solid was dissolved in 13.0 ml ice cold HCl indioxane (12.98 M, 20 equivalents) and left on ice. The progress of thereaction was monitored by TLC (eluent: CHCl₃/MeOH: 9/1). Aftercompletion of the reaction the solvent was removed and the resulting oilwas taken up in methanole and evaporated again. After that the resultingoil was dried over phosphorous-V-oxide and triturated two times withdiethylether.

Yield: 980 mg. The purity was checked by HPLC. The identity of thereaction product was checked by NMR analysis.

Example 2 Chemical Characterization of Selected Dipeptide Compounds

2.1 Melting Point Determination

Melting points were determined on a Kofler heating platform microscopefrom Leica Aktiengesellschaft, the values are not corrected, or on a DSCapparatus (Heumann-Pharma).

2.2 Optical Rotation

The rotation values were recorded at different wavelengths on a“Polarimeter 341” or higher, from the Perkin-Elmer company.

2.3 Measurement Conditions for the Mass Spectroscopy

The mass spectra were recorded by means of electrospray ionisation (ESI)on an “API 165” or “API 365” from the PE Sciex company. The operation iscarried out using an approximate concentration of c=10 μg/ml, thesubstance is taken up in MeOH/H₂O 50:50, 0.1% HCO₂H, the infusion iseffected using a spray pump (20 μl/min). The measurement were made inpositive mode [M+H]⁺, the ESI voltage is U=5600V.

2.4. Results

2.4.1 Tests on Isoleucyl Thiazolidine Fumarate (Isomer)

Substance Mp (° C.) CE (min) MS [α]H₂O L-threo-IT*F 150^(DSC) 160 203−10.7 (405 nm) D-threo-IT*F 147 158 203 not determined L-allo-IT*F 145–6154 203  −4.58 (380 nm) D-allo-IT*F 144–6 150 203    4.5 (380 nm) IT*F =isoleucyl thiazolidine fumarate

The NMR and HPLC data confirm the identity of the substance in question.

2.4.2 Tests on Other Isoleucyl Thiazolidine Salts

IT*salt M (gmol⁻¹) MP (° C.) succinate 522.73 116 tartrate 352.41 122fumarate 520.71 156 hydrochloride 238.77 169 phosphate 300.32 105

Example 3 Synthesis of Xaa-Pro-Yaa Tripeptides

All syntheses were carried out on a peptide synthesizer SP 650 (LabortecAG) applying Fmoc/tBu-strategy. Protected amino acids were purchasedfrom Novabiochem or Bachem. trifluoro acetic acid (TFA) was purchasedfrom Merck, triisopropyl silane (TIS) was purchased from Fluka.

Pre-loaded Fmoc-Yaa-Wang resin (2.8 g/substitution level 0.57 mmol/g)was deprotected using 20% piperidine/N,N-dimethylformamide (DMF). Afterwashing with DMF a solution of 2 eq (1.1 g) of Fmoc-Pro-OH were solvedin DMF (12 ml solvent per gram resin). 2 eq (1.04 g) of2-(1H-Benzotriazole 1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU) and 4 eq (1.11 ml) of N,N-diisopropylethylamine (DIEA) were addedand placed in the reaction vessel. The mixture was shaken at roomtemperature for 20 minutes. Then the coupling cycle was repeated. Aftersubsequent washing with DMF, dichlormethane, isopropanol and diethylether the resulting Fmoc-Pro-IIe-Wang resin was dried and then dividedinto 6 parts before coupling the last amino acid derivative.

Fmoc protecting group was removed as described above. After that 0.54mmol of the Boc-amino acid, 0.54 mmol TBTU and 0.108 mmol DIEA in DMFwere shaken for 20 min. The coupling cycle was repeated. Finally thepeptide resin was washed and dried described above.

The peptide was cleaved from the resin using a mixture oftrifluoroacetic acid (TFA) for 2.5 h, containing the followingscavengers: TFA/H₂O/triisipropylsilane (TIS)=9.5/0.25/0.25

The yields of crude peptides were 80–90% on the average. The crudepeptide was purified by HPLC on a Nucleosil C18 column (7 μm, 250*21.20mm, 100 A) using a linear gradient of 0.1% TFA/H₂O with increasingconcentration of 0.1% TFA/acetonitrile (from 5% to 65% in 40 min) at 6ml/min.

The pure peptide was obtained by lyophilization, identified byElectrospray mass spectrometry and HPLC analysis.

3.1 Results—Identification of Xaa-Pro-Yaa Tripeptides After ChemicalSynthesis

Mass (exp.)¹ Peptide Mass (calc.) [M + H⁺] HPLC k‘² Abu-Pro-Ile 313.4314.0 5.7 Cha-Pro-Ile 381.52 382.0 10.4 Nva-Pro-Ile 327.43 328.2 6.82Phg-Pro-Ile 361.44 362.2 7.9 Nle-Pro-Ile 341.45 342.2 8.09 Pip-Pro-Ile338.56 340.0 6.5 Thr-Pro-Ile 329.4 330.0 5.12 Trp-Pro-Ile 414.51 415.29.85 Phe-Pro-Ile 375.47 376.2 8.96 Ser-Pro-Ile 315.37 316.3 5.24Ser(P)-Pro-Ile 395.37 396.0 3.35 Tyr(P)-Pro-Ile 471.47 472.3 5.14Val-Pro-Val 313.4 314.0 5.07 Ile-Pro-Val 327.43 328.5 6.41Ile-Pro-allo-Ile 341.4 342.0 7.72 Val-Pro-allo-Ile 327.4 328.5 6.51Tyr-Pro-allo-Ile 391.5 392.0 7.02 2-Amino octanoic acid- 369.5 370.210.63 Pro-Ile Ser(Bzl)-Pro-Ile 405.49 406.0 9.87 Orn-Pro-Ile 342.42343.1 3.73 Tic-Pro-Ile 387.46 388.0 8.57 Aze-Pro-Ile 311.4 312.4 5.29Aib-Pro-Ile 313.4 314.0 5.25 t-butyl-Gly-Pro-Ile 341.47 342.1 7.16Ile-Hyp-Ile 356.45 358.2 6.57 t-butyl-Gly-Pro-Val 327.4 328.4 6.32t-butyl-Gly-Pro-Gly 285.4 286.3 3.74 t-butyl-Gly-Pro-Ile-amide 340.47341.3 7.8 t-butyl Gly-Pro-D-Val 327.4 328.6 7.27t-butyl-Gly-Pro-t-butyl-Gly 341.24 342.5 9.09 Ile-Pro-t-butyl-Gly 341.47342.36 6.93 Val-Pro-t-butyl-Gly 327.4 328.15 5.98 ¹[M + H⁺] weredetermined by Electrospray mass spectrometry in positive ionizationmode. ²RP-HPLC conditions: column: LiChrospher 100 RP 18 (5 μm), 125 × 4mm detection (UV): 214 nm gradient system: acetonitrile (ACN)/H₂O (0.1%TFA) from 5% ACN to 50% in 15 min, flow: 1 ml/min

-   k′=(t_(r)−t₀)/t₀-   t₀=1.16 min-   t-butyl-Gly is defined as:

Ser(Bzl) and Ser(P) are defined as benzylserine and phosphorylserine,respectively. Tyr(P) is defined as phosphoryltyrosine.

Example 4 Synthesis of Peptidylketones

Boc-Val-OH (3.00 g, 13.8 mmol) was dissolved in 10 ml of dry THF andcooled down to −15° C. To the mixture CAIBE (1.80 ml, 13.8 mmol) and NMM(1.52 ml, 13.8 mmol) where added and the solution was stirred until theformation of the mixed anhydride was complete. Then the mixture wasbrought to −10° C. and NMM (1.52 ml, 13.8 mmol) was added followed byH-Pro-OMe*HCl (2.29 g, 13.8 mmol). The mixture was allowed to reach roomtemperature and left overnight. After removing the solvent and the usualworkup the resulting ester 1 was taken without further characterisation.The ester 1 was dissolved in HCl/HOAc (5 ml, 6N) and left at 0° C. untilthe removal of the Boc-group was complete. The solvent was then removedand the resulting oil was treated with diethylether to give a whitesolid 2.

Yield: 2.5 g, 80%. Z-Ala-Val-Pro-OMe 3

Z-Ala OH (3.5 g, 15.7 mmol) and 2 (4.18 g, 15.7 mmol) where treated inthe same manner as above for 1, to give 3 as a white solid.

Yield: 4.2 g, 64%. Z-Ala-Val-Pro-OH 4

3 (4.2 g, 9.6 mmol) was dissolved in 30 ml of water/acetone (1/5 v/v)and 11.6 ml NaOH (1N) where added. After completion of the reaction theorganic solvent was removed by evaporation and the resulting solutionwas diluted by 15 ml NaHCO₃ solution (saturated). Then the mixture wasextracted three times by 10 ml of acetic acid ethyl ester. After thatthe solution was brought to pH2 by adding HCl (15% in water). Theresulting mixture was extracted three times by 30 ml of acetic acidethyl ester. The organic layer was separated and washed three times withbrine, dried (Na₂SO₄) and evaporated.

Yield: 3.5 g, 87%. Z-Ala-Val-Pro-CH₂—Br 5

4 (2.00 g, 4.76 mmol) was dissolved in 15 ml of dry THF and convertedinto a mixed anhydride (see compound 1) using CAIBE (0.623 ml, 4.76mmol) and NMM (0.525 ml, 4.76 mmol). The precipitate formed was filteredoff and cooled down to −15° C. Then diazomethane (23.8 mmol in 30 mlether) was dropped into the solution under an argon atmosphere. Afterleaving the mixture for 1 h at 0° C. 1.27 ml of HBr (33% in AcOH) wasadded and the solution was stirred for 30 min at room temperature. Afterthat 70 ml of ether was added and the mixture was washed with 20 ml ofwater. The organic layer was separated and dried (Na₂SO₄) andevaporated.

Yield (crude): 1.8 g, 80%. Z-protected acyloxymethylene ketones

The acid (2 eq) was dissolved in DMF and an equimolar amount of KF wasadded. The suspension was allowed to stir at room temperature for 1hour. Then the brommethylene (1 eq) component was added and the solutionwas allowed to stir overnight. After that the solvent was removed undervacuum and the resulting oil was dissolved in chloroform and washed withbrine. Then the organic layer was separated dried (Na₂SO₄) and thesolvent was removed. The product was purified by column chromatographyusing silica gel and heptane/chloroform.

-   Z-Ala-Val-Pro-CH₂O—C(O)—CH₃ 6-   Acetic acid (230 μl, 4.02 mmol), KF (0.234 g, 4.02 mmol), 5 (1.00 g,    2.01 mmol)

Yield: 0.351 g, 36% Z-Ala-Val-Pro-CH₂O—C(O)-Ph 7

-   Benzoic acid (0.275 g, 2.25 mmol), KF (0.131 mg, 2.25 mmol), 5    (0.56 g. 1.13 mmol)

Yield: 0.34 g, 56% Deprotection

The Z-protected compound was dissolved in HBr/AcOH and stirred. When thereaction was complete ether was added, the white precipitate formed wasfiltered off and dried.

-   H-Ala-Val-Pro-CH2O—C(O)CH₃*HBr 8-   6 (0.351 g, 0.73 mmol)

Yield: 0.252 g, 98% H-Ala-Val-Pro-CH₂O—C(O)Ph*HBr 9

-   7 (0.34 g, 0.63 mmol)

Yield: 0.251 g, 99%

Example 5 Synthesis of Cycloalkylketones

Boc-isoleucinal 2

Oxalylchloride (714 μl, 8.28 mmol) was dissolved in 10 ml of drydichlormethane and brought to −78° C. Then DMSO (817 μl, 8.28 mmol) wasadded dropwise. The solution was stirred for 20 min at −78° C. Then 1(1.00 g, 4.6 mmol) was added and the mixture was stirred for 20 min.After that TEA (2.58 ml, 18.4 mmol) was added and the mixture wasallowed to reach room temperature. The mixture was diluted withhexane/ethylacetate (2/1 v/v) and 10 ml of HCl (10% in water) was added.The organic layer was separated and the aqueous phase was extracted with20 ml of methylenechloride. All organic layers were collected and washedwith brine, followed by water, then dried. The product was purified bycolumn chromatography using silica gel and heptane/chloroform.

Yield: 0.52 g, 52% tert-butylN-1-[cyclopentyl(hydroxy)methyl]-2-methylbutylcarbamate 3

2 (0.52 g, 2.42 mmol) was dissolved in 10 ml of dry THF and cooled downto 0° C. Then cyclopentylmagnesiumbromide (1.45 ml of a 2 M solution)was added. After completion of the reaction (2 ml) of water was addedand solution was neutralized by adding aqueous HCl. Thenmethylenechloride was added and the organic layer was separated anddried (Na₂SO₄). After evaporation the resulting oil was used withoutfurther characterisation.

tert-butyl N-[1-(cyclopentylcarbonyl)-2-methylbutyl]carbamate 4

3 (0.61 g, 2.15 mmol) was treated like 1. Oxalylchloride (333 μl, 3.87mmol), DMSO (382 μl, 5.37 mmol), TEA (1.2 ml, 8.59 mmol)

Yield: 0.180 g, 30% 1-cyclopentyl-3-methyl-1-oxo-2-pentanaminiumchloride 5

4 (0.18 g, 0.63 mmol) was dissolved in 2 ml HCl (7 N in dioxane). Aftercompletion of the reaction the solvent was removed and the resulting oilwas purified by column chromatography on silical gel using achloroform/methanol/water gradient. The resulting oil was trituratedwith ether.

Yield: 0.060 g, 54%

Example 6 Synthesis of Side Chain Modified DPIV-Inhibitors

6.1 Synthesis of Boc-glutamyl-thiazolidine (Boc-Glu-Thia)

Reaction of Boc-Glu(OMe)-OH with Thia*HCl according to Method B (seesection 6.4 for methods), hydrolysis of Boc-Glu(OMe)-Thia according toMethod G

6.1.1 Analytical Data for Boc-Glu-Thia

Empirical formula M_(r) MS [M + H]⁺ Elemental Synthesis TLC: [α]²⁰Danalysis HPLC method R_(f)/system Concentration (calc./ R_(t) CompoundYield m.p. Solvent found) % [min]/system Boc-Glu- C₁₃H₂₂N₂O₅S 319.5 −3.1C: 49.04/48.89 13.93/ Thia 318.38 0.52/A¹ c = 1 H: 6.96/6.82 A² B + G0.42/B¹ methanol N: 8.80/8.59 62% 115–118° C. ¹Thin-layer chromatographySystem A: chloroform/methanol 90:10 System B: benzene/acetone/aceticacid 25:10:0.5 System C: n-butanol/EA/acetic acid/H₂O 1:1:1:1 ²HPLCseparation conditions Column: Nucleosil C-18, 7μ, 250 mm × 21 mm Eluant:isocratic, 40% ACN/water/0.1% TFA Flow rate: 6 ml/min λ = 220 nm6.2 Side Chain-modified Boc-glutamyl Thiazolidines

Boc-Glu-Thia was modified at the γ-carboxylic acid function byintroducing radicals of varying size. The radicals were coupled by wayof their amino group by forming an amide bond to the γ-carboxylic acidfunction, with a variety of coupling methods being used depending on theradical. The following amino components were attached to Boc-Glu-Thiausing the method stated:

Coupling methods Amino component (see section 3.4) Yields Polyethyleneglycol amine (M_(r) ≈ 8000) C 93% H-Gly-Gly-Gly-OH D + E 49%H-Gly-Gly-Gly-Gly-Gly-OH D + E 86%

In 2 cases, purification of the reaction products differs from thegeneral description of synthesis.

Boc-Glu(Gly₅)-Thia

The product already precipitates out from the mixture on stirringovernight; it is subsequently filtered off and washed with 0.1N HCl andcopious amounts of water and then dried over P₄O₁₀ in vacuo.

Boc-Glu(PEG)-Thia

In contrast to the general procedure, the starting materials for thesynthesis are dissolved in a 500-fold excess of DMF. After the reactionis complete, the DMF is completely removed in vacuo and the residue isdissolved in a large amount of methanol. After ether is poured on, toform an upper layer, the product precipitates out together with theunreacted PEG. Fine purification was carried out by preparative HPLCseparation on a gel filtration column (Pharmazia, Sephadex G-25, 90 μm,260 mm–100 mm).

Separating conditions: eluant: water; flow rate: 5 ml/min; λ=220 nm

6.2.2 Synthesis Data for Side Chain-Modified Boc-glutamyl Thiazolidines

Empirical MS [M + H]⁺ Elemental formula TLC/R_(f)/ [α]²⁰D analysis HPLCM_(r) system Concentration (calc./ R_(t) Compound Yield m.p. Solventfound) % [min]/system Boc- C₁₉H₃₁N₅O₈S 490.5 C: 46.62 Glu(Gly₃)- 489.54H: 6.38 Thia 49% N: 14.31 Boc- C₂₃H₃₇N₇O₁₀S 604.5 n.dm. C: 45.76/45.6011.93/A² Glu(Gly₅)- 603.64 0.09/C H: 6.18/6.11 Thia 86% decomp. N:16.24/16.56 from 202° C. Boc- 93% ≈8000 n.dm. n.dm. n.dm. Glu(PEG)-(mass Thia emphasis) 52–53° C. ²HPLC separation conditions Column:Nucleosil C-18, 7μ, 250 mm × 21 mm Eluant: isocratic, 40% ACN/water/0.1%TFA Flow rate: 6 ml/min λ = 220 nm6.3 Side Chain-Modified Glutamyl Thiazolidines

The N-terminal Boc protecting groups were cleaved off the compoundsdescribed in Table 6.2.2 using method F. The substances modified withGly derivatives were purified by preparative HPLC separation and arepresent as trifluoroacetates. The H-Glu(PEG)-Thia was purified on a gelfiltration column in the same manner as the Boc-protected precursor.

6.3.1 Synthesis Data for Side Chain-Modified Glutamyl Thiazolidines

Empirical MS [M + H]⁺ Elemental formula TLC/R_(f)/ [α]²⁰D analysis HPLCM_(r) system Concentration (calc./ R_(t) [min]/ Compound Yield m.p.Solvent found) % system H- C₁₆H₂₄N₅O₈SF₃ 503.45 +4.1 C: 38.17/37.567.84/C³ Glu(Gly₃)- 503.45 0.32/C c = 1 H: 4.80/4.78 Thia *TFA 94% 91–94°C. methanol N: 13.91/13.43 H- C₂₀H₃₀N₇O₁₀SF₃ 617.55 n.dm. C: 38.90/38.828.22/C³ Glu(Gly₅)- 617.55 0.25/C H: 4.90/4.79 Thia *TFA 98% 105–107° C.N: 15.88/15.39 H- 92% ≈8000 n.dm. n.dm. n.dm. Glu(PEG)- (mass Thia *HClemphasis) ³HPLC separation conditions Column: Nucleosil C-18, 7μ, 250 mm× 21 mm Eluant: ACN/water/0.1% TFA Gradient: 20% ACN → 90% ACN over 30min Flow rate: 6 ml/min λ = 220 nm n.dm.—not determined or notdeterminable6.4 General Synthesis ProceduresMethod A: Peptide Bond Attachment by the Mixed Anhydride Method usingCFIBE as Activation Reagent

10 mmol of N-terminally protected amino acid or peptide are dissolved in20 ml of absolute THF. The solution is cooled to −15° C.±2° C. Withstirring in each case, 10 mmol of N-MM and 10 mmol of chloroformic acidisobutyl ester are added in succession, the stated temperature rangebeing strictly adhered to. After approximately 6 min, 10 mmol of theamino component is added. When the amino component is a salt, a further10 mmol of N-MM is then added to the reaction mixture. The reactionmixture is then stirred for 2 h in the cold state and overnight at roomtemperature.

The reaction mixture is concentrated using a rotary evaporator, taken upin EA, washed with 5% KH₂SO₄ solution, saturated NaHCO₃ solution andsaturated NaCl solution and dried over NaSO₄. After removal of thesolvent in vacuo, the compound is recrystallized from EA/pentane.

Method B: Peptide Bond Attachment by the Mixed Anhydride Method usingPivalic Acid Chloride as Activation Reagent

10 mmol of N-terminally protected amino acid or peptide are dissolved in20 ml of absolute THF. The solution is cooled to 0° C. With stirring ineach case, 10 mmol of N-MM and 10 mmol of pivalic acid chloride areadded in succession, the stated temperature range being strictly adheredto. After approximately 6 min, the mixture is cooled to −15° C. and,once the lower temperature has been reached, 10 mmol of the aminocomponent is added. When the amino component is a salt, a further 10mmol of N-MM is then added to the reaction mixture. The reaction mixtureis then stirred for 2 h in the cold state and overnight at roomtemperature.

Further working up is carried out as in Method A.

Method C: Peptide Bond Attachment Using TBTU as Activation Reagent

10 mmol of the N-terminally protected amino acid or peptide and 10 mmolof the C-terminally protected amino component are dissolved in 20 ml ofabsolute DMF. The solution is cooled to 0° C. With stirring in eachcase, 10 mmol of DIPEA and 10 mmol of TBTU are added in succession. Thereaction mixture is stirred for one hour at 0° C. and then overnight atroom temperature. The DMF is completely removed in vacuo and the productis worked up as described in Method A.

Method D: Synthesis of an Active Ester (N-hydroxysuccinimide Ester)

10 mmol of N-terminally protected amino acid or peptide and 10 mmol ofN-hydroxysuccinimide are dissolved in 20 ml of absolute THF. Thesolution is cooled to 0° C. and 10 mmol of dicyclohexylcarbodiimide areadded, with stirring. The reaction mixture is stirred for a further 2 hat 0° C. and then overnight at room temperature. The resultingN,N′-dicyclohexylurea is filtered off and the solvent is removed invacuo and the remaining product is recrystallized from EA/pentane.

Method E: Amide Bond Attachment Using N-hydroxysuccinimide Esters

10 mmol of the C-terminally unprotected amino component is introducedinto an NaHCO₃ solution (20 mmol in 20 ml of water). At room temperatureand with stirring, 10 mmol of the N-terminally protectedN-hydroxysuccinimide ester dissolved in 10 ml of dioxane are slowlyadded dropwise. Stirring of the reaction mixture is continued overnightand the solvent is then removed in vacuo.

Further working up is carried out as in Method A.

Method F: Cleavage of the Boc Protecting Group

3 ml of 1.1N HCl/glacial acetic acid (Method F1) or 3 ml of 1.1NHCl/dioxane (Method F2) or 3 ml of 50% TFA in DCM (Method F3) are addedto 1 mmol of Boc-protected amino acid pyrrolidide, thiazolidide orpeptide. The cleavage at RT is monitored by means of TLC. After thereaction is complete (approximately 2 h), the compound is precipitatedin the form of the hydrochloride using absolute diethyl ether and isisolated with suction and dried over P₄O₁₀ in vacuo. Usingmethanol/ether, the product is recrystallized or reprecipitated.

Method G: Hydrolysis

1 mmol of peptide methyl ester is dissolved in 10 ml of acetone and 11ml of 0.1M NaOH solution and stirred at room temperature. The course ofthe hydrolysis is monitored by means of TLC. After the reaction iscomplete, the acetone is removed in vacuo. The remaining aqueoussolution is acidified, using concentrated KH₂SO₄ solution, until a pH of2–3 is reached. The product is then extracted several times using EA;the combined ethyl acetate fractions are washed with saturated NaClsolution and dried over NaSO₄, and the solvent is removed in vacuo.Crystallization from EA/pentane is carried out.

Example 7 K_(i)-Determination

For K_(i) determination, dipeptidyl peptidase IV from porcine kidneywith a specific activity against glycylprolyl-4-nitroaniline of 37.5U/mg and an enzyme concentration of 1.41 mg/ml in the stock solution wasused.

Assay Mixture:

100 μl test compound in a concentration range of 1*10⁻⁵ M–1*10⁻⁸ Mrespectively were admixed with 50 μl glycylprolyl-4-nitroaniline indifferent concentrations (0.4 mM, 0.2 mM, 0.1 mM, 0.05 mM) and 100 μlHEPES (40 mM, pH7.6; ion strength=0.125). The assay mixture waspre-incubated at 30° C. for 30 min. After pre-incubation, 20 μl DPIV(1:600 diluted) was added and measurement of yellow color developmentdue to 4-nitroaniline release was performed at 30° C. and λ=405 nm for10 min. using a plate reader (HTS7000 plus, Applied Biosystems,Weiterstadt, Germany).

The K_(i)-values were calculated using Graphit version 4.0.13, 4.0.13and 4.0.15 (Erithacus Software, Ltd, UK).

7.1 Results—Ki Values of DPIV Inhibition

Compound Ki [M] H-Asn-pyrrolidine 1.20 * 10⁻⁵ H-Asn-thiazolidine  3.5 *10⁻⁶ H-Asp-pyrrolidine  1.4 * 10⁻⁸ H-Asp-thiazolidine  2.9 * 10⁻⁶H-Asp(NHOH)-pyrrolidine  1.3 * 10⁻⁵ H-Asp(NHOH)-thiazolidine  8.8 * 10⁻⁶H-Glu-pyrrolidine  2.2 * 10⁻⁶ H-Glu-thiazolidine  6.1 * 10⁻⁷H-Glu(NHOH)-pyrrolidine  2.8 * 10⁻⁶ H-Glu(NHOH)-thiazolidine  1.7 * 10⁻⁶H-His-pyrrolidine  3.5 * 10⁻⁶ H-His-thiazolidine  1.8 * 10⁻⁶H-Pro-pyrrolidine  4.1 * 10⁻⁶ H-Pro-thiazolidine  1.2 * 10⁻⁶H-Ile-azididine  3.1 * 10⁻⁶ H-Ile-pyrrolidine  2.1 * 10⁻⁷H-L-threo-Ile-thiazolidine  8.0 * 10⁻⁸ H-L-allo-Ile-thiazolidine  1.9 *10⁻⁷ D-threo-isoleucyl-thiazolidine-fumarate no inhibitionD-allo-isoleucyl-thiazolidine-fumarate no inhibitionH-L-threo-Ile-thiazolidine-succinate  5.1 * 10⁻⁸H-L-threo-Ile-thiazolidine-tartrate  8.3 * 10⁻⁸H-L-threo-Ile-thiazolidine-fumarate  8.3 * 10⁻⁸H-L-threo-Ile-thiazolidine-hydrochloride  7.2 * 10⁻⁸H-L-threo-Ile-thiazolidine-phosphate  1.3 * 10⁻⁷ H-Val-pyrrolidine 4.8 * 10⁻⁷ H-Val-thiazolidine  2.7 * 10⁻⁷ Diprotin A 3.45 * 10⁻⁶Diprotin B 2.24 * 10⁻⁵ Nva-Pro-Ile 6.17 * 10⁻⁶ Cha-Pro-Ile 5.99 * 10⁻⁶Nle-Pro-Ile 9.60 * 10⁻⁶ Phe-Pro-Ile 1.47 * 10⁻⁵ Val-Pro-Val 4.45 * 10⁻⁶Ile-Pro-Val 5.25 * 10⁻⁶ Abu-Pro-Ile 8.75 * 10⁻⁶ Ile-Pro-allo-Ile 5.22 *10⁻⁶ Val-Pro-allo-Ile 9.54 * 10⁻⁶ Tyr-Pro-allo-Ile 1.82 * 10⁻⁵AOA-Pro-Ile 1.26 * 10⁻⁵ t-butyl-Gly-Pro-Ile 3.10 * 10⁻⁶ Ser(Bzl)-Pro-Ile2.16 * 10⁻⁵ Aze-Pro-Ile 2.05 * 10⁻⁵ t-butyl-Gly-Pro-Val 3.08 * 10⁻⁶Gln-Pyrr 2.26 * 10⁻⁶ Gln-Thia 1.21 * 10⁻⁶ Val-Pro-t-butyl-Gly 1.96 *10⁻⁵ t-butyl-Gly-Pro-Gly 1.51 * 10⁻⁵ Ile-Pro-t-butyl-Gly 1.89 * 10⁻⁵t-butyl-Gly-Pro-IleNH₂ 5.60 * 10⁻⁶ t-butyl-Gly-Pro-D-Val 2.65 * 10⁻⁵t-butyl-Gly-Pro-t-butyl-Gly 1.41 * 10⁻⁵ Ile-cyclopentyl ketone 6.29 *10⁻⁶ t-butyl-Gly-cyclohexyl ketone 2.73 * 10⁻⁴ Ile-cyclohexyl ketone5.68 * 10⁻⁵ Val-cyclopentyl ketone 1.31 * 10⁻⁵ Val-Pro-methyl ketone4.76 * 10⁻⁸ Val-Pro-acyloxy methyl ketone 1.05 * 10⁻⁹ Val-Pro-benzoylmethyl ketone  5.36 * 10⁻¹⁰ Val-Pro-benzothiazol methyl ketone 3.73 *10⁻⁸ H-Glu-Thia  6.2 * 10⁻⁷ H-Gly(NHOH)-Thia  1.7 * 10⁻⁶H-Glu(Gly₃)-Thia 1.92 * 10⁻⁸ H-Glu(Gly₅)-Thia 9.93 * 10⁻⁸H-Glu(PEG)-Thia 3.11 * 10⁻⁶t-butyl-Gly is defined as:

Ser(Bzl) and Ser(P) are defined as benzyl-serine and phosphoryl-serine,respectively. Tyr(P) is defined as phosphoryl-tyrosine.

Example 8 Determination of IC₅₀-Values

100 μl inhibitor stock solution were mixed with 100 μl buffer (HEPESpH7.6) and 50 μl substrate (Gly-Pro-pNA, final concentration 0.4 mM) andpreincubated at 30° C. Reaction was started by addition of 20 μlpurified porcine DPIV. Formation of the product pNA was measured at 405nm over 10 min using the HTS 7000Plus plate reader (Perkin Elmer) andslopes were calculated. The final inhibitor concentrations rangedbetween 1 mM and 30 nM. For calculation of IC50 GraFit 4.0.13 (ErithacusSoftware) was used.

8.1 Results—Determination of IC₅₀ Values

Compound IC50 [M] Isoleucyl thiazolidine fumarate 1.28 * 10⁻⁷ Diprotin A4.69 * 10⁻⁶ Diprotin B 5.54 * 10⁻⁵ Phg-Pro-Ile 1.54 * 10⁻⁴ Nva-Pro-Ile2.49 * 10⁻⁵ Cha-Pro-Ile 2.03 * 10⁻⁵ Nle-Pro-Ile 2.19 * 10⁻⁵Ser(P)-Pro-Ile 0.012 Tyr(P)-Pro-Ile 0.002 Phe-Pro-Ile 6.20 * 10⁻⁵Trp-Pro-Ile 3.17 * 10⁻⁴ Ser-Pro-Ile 2.81 * 10⁻⁴ Thr-Pro-Ile 1.00 * 10⁻⁴Val-Pro-Val 1.64 * 10⁻⁵ Ile-Pro-Val 1.52 * 10⁻⁵ Abu-Pro-Ile 3.43 * 10⁻⁵Pip-Pro-Ile 0.100 Ile-Pro-allo-Ile 1.54 * 10⁻⁵ Val-Pro-allo-Ile 1.80 *10⁻⁵ Tyr-Pro-allo-Ile 6.41 * 10⁻⁵ AOA-Pro-Ile 4.21 * 10⁻⁵t-butyl-Gly-Pro-Ile 9.34 * 10⁻⁶ Ser(Bzl)-Pro-Ile 6.78 * 10⁻⁵ Tic-Pro-Ile0.001 Orn-Pro-Ile 2.16 * 10⁻⁴ Gln-Thia 5.27 * 10⁻⁶ Aze-Pro-Ile 7.28 *10⁻⁵ Ile-Hyp-Ile 0.006 t-butyl-Gly-Pro-Val 1.38 * 10⁻⁵ Gln-Pyrr 1.50 *10⁻⁵ Val-Pro-t-butyl-Gly 6.75 * 10⁻⁵ t-butyl-Gly-Pro-Gly 5.63 * 10⁻⁵Ile-Pro-t-butyl-Gly 8.23 * 10⁻⁵ t-butyl-Gly-Pro-IleNH₂ 2.29 * 10⁻⁵t-butyl-Gly-Pro-D-Val 1.12 * 10⁻⁴ t-butyl-Gly-Pro-t-butyl-Gly 2.45 *10⁻⁵ Aib-Pro-Ile no inhibition Ile-cyclopentyl ketone 3.82 * 10⁻⁵t-butyl-Gly-cyclohexyl ketone 2.73 * 10⁻⁴ Ile-cyclohexyl ketone 2.93 *10⁻⁴ Val-cyclopentyl ketone 4.90 * 10⁻⁵ Val-cyclohexyl ketone 0.001Val-Pro-methyl ketone 5.79 * 10⁻⁷ Val-Pro-acyloxy methyl ketone 1.02 *10⁻⁸ Val-Pro-benzoyl methyl ketone 1.79 * 10⁻⁸ Val-Pro-benzothiazolmethyl ketone 1.38 * 10⁻⁷t-butyl-Gly is defined as:

Ser(Bzl) and Ser(P) are defined as benzyl-serine and phosphoryl-serine,respectively. Tyr(P) is defined as phosphoryl-tyrosine.

Example 9 Inhibition of DPIV-Like Enzymes—Dipeptidyl Peptidase II

DP II (3.4.14.2) releases N-terminal dipeptides from oligopeptides ifthe N-terminus is not protonated (McDonald, J. K., Ellis, S. & Reilly,T. J., 1966, J. Biol. Chem., 241, 1494–1501). Pro and Ala in P₁-positionare preferred residues. The enzyme activity is described as DPIV-likeactivity, but DP II has an acidic pH-optimum. The enzyme used waspurified from porcine kidney.

Assay:

100 μl glutaminyl pyrrolidine or glutaminyl thiazolidine in anconcentration range of 1*10⁻⁴ M–5*10⁻⁸ M were admixed with 100 μl μlbuffer solution (40 mM HEPES, pH7.6, 0.015% Brij, 1 mM DTT), 50 μllysylalanylaminomethylcoumarine solution (5 mM) and 20 μl porcine DP II(250fold diluted in buffer solution). Fluorescence measurement wasperformed at 30° C. and λ_(exiatation)=380 nm, λ_(emission)=465 nm for25 min using a plate reader (HTS7000plus, Applied Biosystems,Weiterstadt, Germany). The K_(i)-values were calculated using Graphit4.0.15 (Erithacus Software, Ltd., UK) and were determined asK_(i)=8.52*10⁻⁵ M±6.33*10⁻⁶ M for glutaminyl pyrrolidine andK_(i)=1.07*10⁻⁵ M±3.81*10⁻⁷ M for glutaminyl thiazolidine.

Example 10 Cross Reacting Enzymes

Glutaminyl pyrrolidine and glutaminyl thiazolidine were tested for theircross reacting potency against dipeptidyl peptidase I, prolyloligopeptidase and prolidase.

Dipeptidyl Peptidase I (DP I, Cathepsin C):

DP I or cathepsin C is a lysosomal cysteine protease which cleaves offdipeptides from the N-terminus of their substrates (Gutman, H. R. &Fruton, J. S., 1948, J. Biol: Chem., 174, 851–858). It is classified asa cysteine protease. The enzyme used was purchased from Qiagen (QiagenGmbH, Hilden, Germany). In order to get a fully active enzyme, theenzyme was diluted 1000fold in MES buffer pH5.6 (40 mM MES, 4 mM DTT, 4mM KCl, 2 mM EDTA, 0.015% Brij) and pre-incubated for 30 min at 30° C.

Assay:

50 μl glutaminyl pyrrolidine or glutaminyl thiazolidine in aconcentration range of 1*10⁻⁵ M–1*10⁻⁷ M were admixed with 110 μlbuffer-enzyme-mixture. The assay mixture was pre-incubated at 30° C. for15 min. After pre-incubation, 100 μl histidylseryl-β-nitroaniline(2*10⁻⁵M) was added and measurement of yellow color development due toβ-nitroaniline release was performed at 30° C. and λ_(excitation)=380nm, λ_(emission)=465 nm for 10 min., using a plate reader (HTS7000 plus,Applied Biosystems, Weiterstadt, Germany).

The IC₅₀-values were calculated using Graphit 4.0.15 (ErithacusSoftware, Ltd., UK). No inhibition of the DP I enzyme activity byglutaminyl pyrrolidine or glutaminyl thiazolidine was found.

Prolyl Oligopeptidase (POP)

Prolyl oligopeptidase (EC 3.4.21.26) is a serine type endoprotease whichcleaves off peptides at the N-terminal part of the Xaa-Pro bond (Walter,R., Shlank, H., Glass, J. D., Schwartz, I. L. & Kerenyi, T. D., 1971,Science, 173, 827–829). Substrates are peptides with a molecular weightup to 3000 Da. The enzyme used was a recombinant human prolyloligopeptidase. Recombinant expression was performed in E. coli understandard conditions as described elsewhere in the state of the art.

Assay:

100 μl glutaminyl pyrrolidine or glutaminyl thiazolidine in anconcentration range of 1*10⁻⁴ M–5*10⁻⁸ M were admixed with 100 μl μlbuffer solution (40 mM HEPES, pH7.6, 0.015% Brij, 1 mM DTT) and 20 μlPOP solution. The assay mixture was pre-incubated at 30° C. for 15 min.After pre-incubation, 50 μl glycylprolylprolyl-4-nitroaniline solution(0.29 mM) was added and measurement of yellow color development due to4-nitroaniline release was performed at 30° C. and λ=405 nm for 10 minusing a plate reader (sunrise, Tecan, Crailsheim, Germany). TheIC₅₀-values were calculated using Graphit 4.0.15 (Erithacus Software,Ltd., UK). No inhibition of POP activity by glutaminyl pyrrolidine orglutaminyl thiazolidine was found.

Prolidase (X-Pro Dipeptidase)

Prolidase (EC 3.4.13.9) was first described by Bergmann & Fruton(Bergmann, M. & Fruton, J S, 1937, J. Biol. Chem. 189–202). Prolidasereleases the N-terminal amino acid from Xaa-Pro dipeptides and has a pHoptimum between 6 and 9.

Prolidase from porcine kidney (ICN Biomedicals, Eschwege, Germany) wassolved (1 mg/ml) in assay buffer (20 mM NH₄(CH₃COO)₂, 3 mM MnCl₂, pH7.6). In order to get a fully active enzyme the solution was incubatedfor 60 min at room temperature.

Assay:

450 μl glutaminyl pyrrolidine or glutaminyl thiazolidine in anconcentration range of 5*10⁻³ M–5*10⁻⁷ M were admixed with 500 μl buffersolution (20 mM NH₄(CH₃COO)₂, pH 7.6) and 250 μl IIe-Pro-OH (0.5 mM inthe assay mixture). The assay mixture was pre-incubated at 30° C. for 5min. After pre-incubation, 75 μl Prolidase (1:10 diluted in assaybuffer) were added and measurement was performed at 30° C. and λ=220 nmfor 20 min using a UV/Vis photometer, UV1 (Thermo Spectronic, Cambridge,UK).

The IC 50-values were calculated using Graphit 4.0.15 (ErithacusSoftware, Ltd., UK). They were determined as IC₅₀>3 mM for glutaminylthiazolidine and as IC₅₀=3.4*10⁴M±5.63*10⁻⁵ for glutaminyl pyrrolidine.

Example 11 Determination of DPIV Inhibiting Activity After Intravasaland Oral Administration to Wistar Rats

Animals

Male Wistar rats (Shoe: Wist(Sho)) with a body weight ranging between250 and 350 g were purchased from Tierzucht Schönwalde (Schönwalde,Germany).

Housing Conditions

Animals were single-caged under conventional conditions with controlledtemperature (22±2° C.) on a 12/12 hours light/dark cycle (light on at06:00 AM). Standard pelleted chow (ssniff® Soest, Germany) and tap wateracidified with HCl were allowed ad libitum.

Catheter Insertion into Carotid Artery

After ≧one week of adaptation at the housing conditions, catheters wereimplanted into the carotid artery of Wistar rats under generalanaesthesia (i.p. injection of 0.25 ml/kg b.w. Rompun® [2%], BayerVital,Germany and 0.5 ml/kg b.w. Ketamin 10, Atarost GmbH & Co., Twistringen,Germany). The animals were allowed to recover for one week. Thecatheters were flushed with heparin-saline (100 IU/ml) three times perweek. In case of catheter dysfunction, a second catheter was insertedinto the contra-lateral carotid artery of the respective rat. After oneweek of recovery from surgery, this animal was reintegrated into thestudy. In case of dysfunction of the second catheter, the animal waswithdrawn from the study. A new animal was recruited and the experimentswere continued in the planned sequence, beginning at least 7 days aftercatheter implantation.

Experimental Design

Rats with intact catheter function were administered placebo (1 mlsaline, 0.154 mol/l) or test compound via the oral and the intra-vasal(intra-arterial) route. After overnight fasting, 100 μl samples ofheparinised arterial blood were collected at −30, −5, and 0 min. Thetest substance was dissolved freshly in 1.0 ml saline (0.154 mol/l) andwas administered at 0 min either orally via a feeding tube (75 mm; FineScience Tools, Heidelberg, Germany) or via the intra-vasal route. In thecase of oral administration, an additional volume of 1 ml saline wasinjected into the arterial catheter. In the case of intra-arterialadministration, the catheter was immediately flushed with 30 μl salineand an additional 1 ml of saline was given orally via the feeding tube.

After application of placebo or the test substances, arterial bloodsamples were taken at 2.5, 5, 7.5, 10, 15, 20, 40, 60 and 120 min fromthe carotid catheter of the conscious unrestrained rats. All bloodsamples were collected into ice cooled Eppendorf tubes(Eppendorf-Netheler-Hinz, Hamburg, Germany) filled with 10 μl 1M sodiumcitrate buffer (pH 3.0) for plasma DPIV activity measurement. Eppendorftubes were centrifuged immediately (12000 rpm for 2 min, HettichZentrifuge EBA 12, Tuttlingen; Germany): The plasma fractions werestored on ice until analysis or were frozen at −20° C. until analysis.All plasma samples were labelled with the following data:

-   -   Code number    -   Animal Number    -   Date of sampling    -   Time of sampling        Analytical Methods

The assay mixture for determination of plasma DPIV activity consisted of80 μl reagent and 20 μl plasma sample. Kinetic measurement of theformation of the yellow product 4-nitroaniline from the substrateglycylprolyl-4-nitroaniline was performed at 390 nm for 1 min at 30° C.after 2 min pre-incubation at the same temperature. The DPIV activitywas expressed in mU/ml.

Statistical Methods

Statistical evaluations and graphics were performed with PRISM® 3.02(GraphPad Software, Inc.). All parameters were analysed in a descriptivemanner including mean and SD.

11.1 Results—In Vivo DPIV-Inhibition at t_(max)

Dose STRUCTURE (mg/kg) i.v. (%) p.o. (%) Gln-Pyrr 100 80 67 Gln-Thia 10088 71 Diprotin A 100 73 no inhibition Diprotin B 100 50 no inhibitionTyr(P)-Pro-Ile 100 37 no inhibition t-butyl-Gly-Pro-Ile 100 71 28t-butyl-Gly-Pro-Val 100 72 25 Ala-Val-Pro-acyloxy methyl 100 89 86ketone Ala-Val-Pro-benzoyl- 100 97 76 methyl ketone Ile-cyclopentylketone 100 34 15

Example 12 Action of Side Chain-Modified Glutamyl Thiazolidines asNon-Readily-Transportable DPIV-Inhibitors

Side chain-modified glutamyl thiazolidines having a structureH-Glu(X)-Thia were synthesised, with polyethylene glycol or glycineoligomers of various chain lengths being used as X (see Method A ofexample for description of synthesis). The binding characteristics ofthose derivatives and their transportability by the peptide transporterPepT1 were investigated.

Surprisingly, it was found that the side chain modifications alter thebinding characteristics of the compounds to DPIV only to a slightextent. In contrast, the ability of the inhibitors to be transported bythe peptide transporter is dramatically diminished by the side chainmodification.

Side chain modified inhibitors of DPIV or DPIV-like enzymes aretherefore well suited to achieving site directed inhibition of DPIV inthe body.

12.1 Results: Transportability of Selected DPIV-Inhibitors.

Compound EC50 (mM)¹ I_(max) (nA)² amino acid thiazolidines H-Ile-Thia0.98 25 ± 8  H-Glu-Thia 1.1 35 ± 13 side chain-modifiedglutamylthiazolidines H-Gly(NHOH)-Thia 3.18 42 ± 11 H-Glu(Gly₃)-Thia8.54 n.d.³ H-Glu(Gly₅)-Thia >10 n.d.³ H-Glu(PEG)-Thia >10 n.d.³¹Effective concentrations of the compounds inhibiting the binding of³H-D-Phe-Ala (80 mM) to PepT1-expressing P. pastoris cells by 50% (EC₅₀values) ²Transport characteristics at PepT1-expressing oocytes of X.leavis - by means of two-electrode voltage clamp method, I = inwardcurrents generated by the transport

Example 13 In vivo Cancer Cell Adhesion Assay

Using a novel in vivo adhesion assay which takes advantage of vital dyelabeled tumor cells and their detection in the target tissue in situ(von Hörsten et al, 2000; Shingu et al., 2002), the current exampleinvestigates whether the in vivo adhesion of MADB106 tumor cells differsin DPIV in treated wild type F344 rats and F344 substrains with amutation of the DPIV gene.

Animals, Injection of Tumor Cells and Processing of Lungs

F344USA, F344JAP and F344GER substrains were obtained from a breedingcolony at the Central Animal Laboratory at Hannover Medical School,Germany. All substrains were bred for one generation and maintained in aspecific-pathogen-free facility at 25° C. under a 12 h light–12 h darkcycle (light on at 07.00 h), with ad libitum access to food and water.The exact number of animals used per experiment is indicated by the Fvalues with at least four animals per condition and time point.

Cell culture, injection of tumor cells, dissection of the animals andimmunohistochemistry were conducted as previously described (von Hörstenet al., 2000). In brief, 1×10⁶ MADB106 tumor cells derived from logphase of tumor growth were injected via the lateral tail vein and lungsremoved at different time points thereafter. For in situ quantificationof tumor cells at early time points after injection (30 min), cells werevital dye stained using the fluorescein derivate 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) beforeinjection. For quantification of lung surface colonies at later timepoints (2 weeks after tumor cell inoculation), en-bloc dissected lungsand the heart were injected with 8 ml Bouin's solution (72% saturatedpicric acid solution, 23% formaldehyde, and 5% glacial acetic acid) andfixed in the same solution until lung surface nodules were counted (seebelow).

Experiments

Four experiments were conducted:

Effect of a single injection of isoleucyl thiazolidine fumarate (2 mgi.v.+isoleucyl thiazolidine fumarate 2 mg i.p.) on lung tumorcolonization in F344USA wild-type rats;

Effect of single injection of isoleucyl thiazolidine fumarate 2 mgi.v.+isoleucyl thiazolidine fumarate 2 mg i.p. on tumor cell adhesion tolungs of F344JAP, F344GER and F344USA rats;

Effect of single injection of isoleucyl cyanopyrrolidine (0.1 mgi.v.+0.1 mg i.p.) on tumor cell adhesion to lungs of F344USA wild-typerats;

Effect of single injection of valyl-pyrrolidine fumarate (0.1 mgi.v.+0.1 mg i.p.) on tumor cell adhesion to lungs of F344USA wild-typerats;

Immunohistochemistry of CFSE-Labeled Tumor Cells in Lungs

Immunostaining of CFSE-labeled MADB106 tumor cells was achieved usingmAb characterizing the intracellular CFSE antigen (anti-CFSE; mAb DE1,Boehringer, Mannheim, Germany; mouse, 1:100). For immunohistochemistry,one or two consecutive APAAP stainings were performed as previouslydescribed (von Hörsten et al, 2000; Shingu et al., 2002). Controlsections were included in which one or both primary antibodies wereomitted.

Quantification of Tumor Targets: In Vivo/In Situ Cell Adhesion Assay

Vital dye (Carboxyfluorescein; CFSE) labeling of MADB106 tumor cellsallows the quantification of tumor cells and NK cells in thick sectionsof lung tissue by stereology in situ (von Hörsten et al, 2000). In thepresent study we produced thin sections (8 μm) of the same lungs (n=10)and performed additional microscopic counting by image analysis of DE1positive cells. This was done to further simplify the previouslyvalidated stereological quantification technique. Therefore, in thepresent study, the assessment of DE1 positive tumor cells in lung tissuefrom different substrains 30 min after tumor inoculation was carried outusing image analysis approach. All CFSE-labeled MADB106 tumor cells andleukocyte subsets within a grid on the ocular lens were counted (ZeissKpl-W 12.5×; grid 0.75×0.75 mm=0.5625 mm²/grid, using a Zeiss Neofluarobjective, ×10, NA=0.3). Each right upper lobe of the lungs wassectioned at 6 randomly chosen non-adjacent levels. From each level,three sections were evaluated. On average, 30 grid numbers per sectionwere examined (i.e. 0.5625 mm²/grid×30 grids×3 sections×6 levels)resulting in an area per animal of 3.04 cm².

Quantification of Macrometastasis on Lungs

For quantification of lung surface colonies at later time points (2weeks after tumor cell inoculation), en-bloc dissected lungs and theheart were injected with 8 ml Bouin's solution (72% saturated picricacid solution, 23% formaldehyde, and 5% glacial acetic acid) and fixedin the same solution until lung surface nodules were counted. Threeareas per lungs were examined using a gauge (1 cm²) and lung surfacecolony numbers were expressed as mean/cm² according to the method ofWexler (Wexler, 1966).

Statistical Analysis

Data from in vivo adhesion assay were analyzed by one-way ANOVAs andFisher's PLSD post hoc tests, if appropriate. An asterisk indicatessignificant post hoc effects vs. saline (SHAM) treated controls obtainedby Fisher's PLSD. All data are presented as means±S.E.M.

Results

Effect of a Single Injection of Isoleucyl Thiazolidine Fumarate on LungTumor Colonization in F344USA Rats

The number of lung surface tumor colonies after single isoleucylthiazolidine fumarate administration in F344 rats 2 weeks afterinjection of MADB106 tumor cells is illustrated in FIG. 1. One factorANOVA revealed no significant effect (F(1,12)=3.2; p=0.1 n.s.) on colonynumbers. A trend toward decreased colony numbers in experimental ratswas evident.

Effect of Single Injection of Isoleucyl Thiazolidine Fumarate on TumorAdhesion in F344JAP, F344GER and F344USA Rats

The mean number of CFSE positive cells in lung tissue at 30 min afterinoculation of MADB106 tumor cells in the three substrains isillustrated in FIG. 2. Two-way ANOVA showed a significant effect of“substrain” (F(2,43)=3.5; p<0.04) and “treatment” (F(1,43)=44.1;p<0.0001), as well as a significant interaction factors but nosignificant interaction (F(2,43)=26.2; p<0.0001). Separate one factorANOVAs split for these substrains revealed that these effects aresignificant.

Effect of Single Injection of Isoleucyl Cyanopyrrolidine TFA on TumorAdhesion of F344USA Rats

The mean number of CFSE positive cells in lung tissue at 30 min afterinoculation of MADB106 tumor cells after isoleucyl cyanopyrrolidine TFAtreatment is illustrated in FIG. 3. ANOVA showed no significant effectof “treatment” (F(1,18)=0.1; p=0.8 n.s.).

Effect of Single Injection of Valyl Pyrrolidine Fumarate on TumorAdhesion of F344USA Rats

The mean number of CFSE positive cells in lung tissue at 30 min afterinoculation of MADB106 tumor cells after valyl pyrrolidine fumaratetreatment is illustrated in FIG. 4. ANOVA showed no significant effectof “treatment” (F(1,18)=0.6; p=0.5 n.s.).

Discussion

Tumor cell adhesion and colonization is significantly modified by singleinjection of isoleucyl thiazolidine fumarate only in mutant F344substrains suggesting an interaction of the ligand with mutant DPIV andtumor cells. Since the ligand did not significantly affect tumoradhesion in wild type F344USA rats, this may indicate that compound isnot interacting with the binding site of MADB106 tumor cells.

Example 14 Cancer Colonization Assays

In the previous example it was demonstrated that MADB106 tumor celladhesion is significantly modified by a single administration ofisoleucyl thiazolidine fumarate only in mutant F344 substrains but notin wild type DPIV expressing F344USA rats. DPIV inhibitors/ligands mayinteract with the growth of tumor metastases exhibiting propertiessimilar to chemotherapeutic compounds and/or immunotherapeuticalcompounds. In contrast to that, the current example investigates whetherthe tumor colonization of MADB106 tumor cells differs in chronicallyDPIV-inhibitor treated wild type F344 rats.

Animals, Injection of Tumor Cells and Processing of Lungs

F344USA rats were obtained from a breeding colony at the Central AnimalLaboratory at Hannover Medical School, Germany. All rats were bred atleast for one generation and maintained in a specific-pathogen-freefacility at 25° C. under a 12 h light–12h dark cycle (light on at 07.00h), with ad libitum access to food and water. The exact number ofanimals used per experiment is indicated by the F values with at leastfour animals per condition and time point.

Cell culture, injection of tumor cells, dissection of the animals andimmunohistochemistry were conducted as previously described (von Hörstenet al., 2000; Shingu et al., 2002). In brief, 1×10⁶ MADB106 tumor cellsderived from log phase of tumor growth were injected via the lateraltail vein and lungs removed at different time points thereafter. For insitu quantification of tumor cells at early time points after injection(30 min), cells were vital dye stained using the fluorescein derivate5-(and -6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) beforeinjection. For quantification of lung surface colonies at later timepoints (2 weeks after tumor cell inoculation), en-bloc dissected lungsand the heart were injected with 8 ml Bouin's solution (72% saturatedpicric acid solution, 23% formaldehyde, and 5% glacial acetic acid) andfixed in the same solution until lung surface nodules were counted (seebelow).

Experiments

Two experiments were conducted:

Effect of chronic infusion of different dosages of isoleucylthiazolidine fumarate (0 mg, 0.04 mg, 0.4 mg, 4 mg/24 h intragastral viaimplanted osmotic minipumps) on body weight change and lung tumorcolonization in F344USA wild-type rats

Effect of chronic infusion of isoleucyl thiazolidine fumarate (4 mg/24 hintragastral via implanted osmotic minipumps), lisoleucylcyanopyrrolidine TFA (0.1 mg/24 h intragastral via implanted osmoticminipumps) and valyl pyrrolidine fumarate (0.1 mg/24 h intragastral viaimplanted osmotic minipumps) on lung tumor colonization in F344USAwild-type rats.

Implantation of Osmotic Minipumps for Chronic Intragastric Infusion ofCompounds

Osmotic minipumps (Alzet model 2ML4; flow rate, 2.5 μl/hr; AlzaCorporation), administering a constant supply of the differentcompounds, aseptically prefilled with either saline or DPIV inhibitorwere placed subcutaneously in the abdominal area. Minipums were attachedto a cannula via polyethylene tubing. The cannula was implantedintragastrically with a heating-induced enlarged tip of the cannula inthe lumen of the gaster.

Quantification of Macrometastasis on Lungs

For quantification of lung surface colonies at later time points (2weeks after tumor cell inoculation), en-bloc dissected lungs and theheart were injected with 8 ml Bouin's solution (72% saturated picricacid solution, 23% formaldehyde, and 5% glacial acetic acid) and fixedin the same solution until lung surface nodules were counted. Threeareas per lungs were examined using a gauge (1 cm²) and lung surfacecolony numbers were expressed as mean/cm² according to the method ofWexler (Wexler, 1966).

Statistical Analysis

Data from in vivo body weight gain and number of lung surface tumorcolonies were analyzed by one-way ANOVAs and Fisher's PLSD post hoctests, if appropriate. An asterisk indicates significant post hoceffects vs. saline (SHAM) treated controls obtained by Fisher's PLSD.All data are presented as means±S.E.M.

Results

Effect of Chronic Infusion of Isoleucyl Thiazolidine Fumarate (0 mg,0.04 mg, 0.4 mg, 4 mg/24 h) on Lung Tumor Colonization

The change of body weight after chronic infusion of different dosages ofisoleucyl thiazolidine fumarate in F344 rats 2 weeks after injection ofMADB106 tumor cells is illustrated in FIG. 5. One factor ANOVA revealeda significant effect (F(3,22)=3.5; p=0.03) on body weight, which becamesignificant in the post-hoc analysis at the 0.4 mg and 4 mg dosages.

The number of lung surface tumor colonies after chronic infusion ofdifferent dosages of isoleucyl thiazolidine fumarate in F344 rats 2weeks after injection of MADB106 tumor cells is illustrated in FIG. 6.One factor ANOVA revealed a significant effect (F(3,22)=3.8; p=0.03) oncolony numbers, which became significant in the post-hoc analysis at the4 mg dosage.

Effect of Chronic Infusion of Isoleucyl Thiazolidine Fumarate, IsoleucylCyanopyrrolidine TFA, and Valyl Pyrrolidine Fumarate on Lung TumorColonization

The number of lung surface tumor colonies after chronic infusion ofisoleucyl thiazolidine fumarate; isoleucyl cyanopyrrolidine TFA, andvalyl pyrrolidine fumarate in F344 rats 2 weeks after injection ofMADB106 tumor cells is illustrated in FIG. 7. One factor ANOVA revealeda significant effect (F(3,20)=3.8; p=0.03) on colony numbers, whichbecame significant in the post-hoc analysis for isoleucylcyanopyrrolidine TFA and isoleucyl thiazolidine fumarate compounds.

Discussion

Metastasis of MADB106 is reduced by chronic treatment using differentDPIV Inhibitors (isoleucyl thiazolidine fumarate; isoleucylcyanopyrrolidine TFA) suggesting protective-like class effects.Possibly, isoleucyl thiazolidine fumarate and isoleucyl cyanopyrrolidineTFA protect from metastasis either via interaction with cell adhesionprocesses or via a modification of the cellular host defense mechanisms.It is also possible that DPIV inhibitor treatment exhibits cytostaticeffects. These antimetastic effects substantiate the biologicalproperties of DPIV Inhibitors for the treatment of cancer and metastaticdisease.

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1. A method for treating breast cancer, the method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of at least one inhibitor of dipeptidyl peptidase IV (DPIV),wherein said inhibitor is an amino acid linked to a thiazolidine or apyrrolidine group by a peptide bond.
 2. The method according to claim 1,wherein treating comprises treating tumor cell metastasis.
 3. The methodaccording to claim 1, wherein treating comorises treating tumorcolonization.
 4. The method according to claim 1, wherein the at leastone inhibitor is selected from the group consisting of L-threo-isoleucylpyrrolidine, L-allo-isoleucyl thiazolidine, L-allo-isoleucylpyrrolydine, L-glutaminyl thiazolidine, L-glutaminyl pyrrolidine,L-glutamic acid thiazolidine, L-glutamic acid pyrrolidine and saltsthereof.
 5. A method according to claim 1 wherein the amino acid isselected from the group consisting of leucine, valine, glutamine,glutamic acid, proline, isoleucine, asparagine and aspartic acid.